Process for selectively producing 1-phosphorylated sugar derivative anomer and process for producing nucleoside

ABSTRACT

A desired isomer is selectively prepared by phosphorolyzing and isomerizing an anomer mixture of a 1-phosphorylated saccharide derivative while crystallizing one of the isomers to displace the equilibrium. Furthermore, using the action of a nucleoside phosphorylase, a nucleoside is prepared from the 1-phosphorylated saccharide derivative obtained and a base with improved stereoselectivity and a higher yield. This process is an anomer-selective process for preparing a 1-phosphorylated saccharide derivative and a nucleoside.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a process for producing a1-phosphorylated saccharide derivative. 1-phosphorylated saccharidesare—widely distributed in the living world, are reaction substrates fora variety of enzymes and are utilized starting materials for preparinguseful substances such as drugs and nutritional foods. Synthetic1-phosphorylated saccharide derivatives have been expected to be used asstarting materials for preparing drugs such as antiviral agents andenzyme inhibitors.

[0003] This invention also relates to a process for producing anucleoside compound used as a starting material or drug substance formedical drugs such as antiviral, anticancer and antisense drugs.

[0004] 2. Description of the Prior Art

[0005] There are known processes for producing a 1-phosphorylatedsaccharide such as:

[0006] 1) condensation of a 1-bromosaccharide with a silver phosphatesalt (J. Biol. Chem., Vol.121, p.465 (1937); J. Am. Chem. Soc., Vol.78,p.811 (1956); J. Am. Chem. Soc., Vol.79, p.5057 (1957));

[0007] 2) condensation of a 1-halogenated saccharide with atriethylamine salt of dibenzylphosphoric acid (J. Am. Chem. Soc.,Vol.77, p.3423 (1955); J. Am. Chem. Soc., Vol.80, p.1994 (1958); J. Am.Chem. Soc., Vol.106, p.7851 (1984); J. Org. Chem., Vol.59, p.690(1994));

[0008] 3) thermal condensation of a 1-acetylated saccharide withorthophosphoric acid (J. Org. Chem., Vol.27, p.1107 (1962); CarbohydrateRes., Vol.3, p.117 (1966); Carbohydrate Res., Vol.3, p.463 (1967); Can.J. Biochem., Vol.50, p.574 (1972));

[0009] 4) condensation of dibenzylphoshoric acid with a saccharideactivated at 1-position by imidation (Carbohydrate Res., Vol.61, p.181(1978); Tetrahedron Lett., Vol.23, p.405 (1982));

[0010] 5) treatment of a saccharide activated at 1-position by thalliumor lithium alcolate with dibenzylphosphoric chloride (Carbohydrate Res.,Vol.94, p.165 (1981); Chem. Lett., Vol.23, p.405 (1982));

[0011] 6) phosphorolysis of a nucleoside utilizing action of nucleosidephosphorylase to form a 1-phoshorylated saccharide derivative (J. Biol.Chem., Vol.184, p.437 (1980)).

[0012] These processes have the following drawbacks.

[0013] A common problem in the chemical processes described in theabove 1) to 5) is that it is difficult to establish a general syntheticmethod for preparing a desired isomer with a good selectivity due tovariation in an anomer selectivity between α/β anomers owing toinfluence of a functional group adjacent to 1-position. For achievingselectivity and a good yield, the presence of 2-acetoxy or acetaminogroup is essential. However, since 2-deoxysaccharide is unstable, thesesynthetic processes may be limited to a considerably narrow applicationrange. Thus, it is difficult to control anomer selectivity so thatcolumn chromatography purification is required, leading to a poor yield(Chem. Zvesti, Vol.28(1), p.115 (1974); Izv. Akad. Nauk SSSR, Ser.Khim., Vol.8, p.1843 (1975)).

[0014] Of course, there have been no reports for chemical preparation ofa 1-phosphorylated 2-deoxyfuranose which is more unstable than a1-phosphorylated 2-deoxypyranose, resulting in more difficultselectivity control.

[0015] In terms of 6), preparation of a nucleoside itself is difficultexcept a quite limited type of rebonucleosides such as inosine. Alimited type of 1-phosphorylated saccharide derivatives such asribose-1-phosphate can be, therefore, prepared. In addition, since anucleoside itself as a starting material is expensive, the process isnot satisfactory in its cost.

[0016] As described above, the term “nucleoside phosphorylase” is ageneric name for enzymes capable of phosphorolysis of an N-glycosidebond in a nucleoside in the presence of phosphoric acid, which catalyzea reaction represented by the following equation:

Nucleoside+Phosphoric acid (salt)→Base+1-Phosphorylated saccharidederivative

[0017] The enzymes which may be generally categorized into two groups ofpurine nucleoside phosphorylases and pyrimidine nucleosidephosphorylases, are widely distributed in the living world; they arepresent in tissues of mammals, birds and fish; yeasts; and bacteria. Theenzyme reaction is reversible and there have been disclosed methods forsynthesis of a variety of nucleosides utilizing a reverse reaction; forsyntheses of thymidine (thymine, adenine or guanine) (JP-A 01-104190),2′-deoxyadenosine (JP-A 11-137290) or 2′-deoxyguanosine (JP-A 11-137290)from 2′-deoxyribose 1-phosphate and a nucleic-acid base.

[0018] Furthermore, Agric. Biol. Chem., Vol.50 (1), pp.121-126 (1986)has described a process where by a reaction using a purine nucleosidephosphorylase from Enterobacter aerogenes in the presence of phosphoricacid, inosine is decomposed into ribose 1-phosphate and hypoxanthine andthe former isolated using an ion-exchange resin and1,2,4-triazole-3-carboxamide are also treated with a purine nucleosidephosphorylase from Enterobacter aerogenes to prepare ribavirin as anantiviral agent.

[0019] However, as described above, an industrial process for producinga 1-phosphorylated saccharide derivative has not been established, andthus an industrial process for preparation of a universally usefulnucleoside utilizing a reverse reaction of a nucleoside phosphorylasehas been also not established.

[0020] Furthermore, since the reaction for forming a nucleoside from1-phosphorylated saccharide derivative and a base utilizing the reversereaction of the enzyme is reversible, there is a technical drawback thatan inversion rate cannot be improved.

SUMMARY OF THE INVENTION

[0021] An objective of this invention is to provide a highly universaland anomer-selective process for preparing 1-phospholyrated saccharidederivative which is not influenced by difference in a saccharideskeleton such as furanose and pyranose, presence of a substituent suchas a deoxysaccharide or a saccharide type, i.e., natural or synthetic.

[0022] Another objective of this invention is to provide a highlyuniversal process for producing a nucleoside by treating a1-phosphorylated saccharide derivative and a nucleic-acid base with anucleoside phosphorylase and a method for improving an inversion ratefor the nucleoside in the reaction.

[0023] In other words, the ultimate objective of this invention is toprovide a process for producing a highly pure nucleoside with a lowercost by achieving the first and the second objectives above.

[0024] We have intensely made attempts for achieving the firstobjective. Finally, we have found that a 1-phosphorylated saccharidederivative is present in an equilibrium with an anomer and a dimer ofthe 1-pohsphorylated saccharide derivative under certain conditions andthat the conditions may be adjusted to allow only a desired anomer to beprecipitated as crystals so that the equilibrium may be displaced towardthe preferable direction to provide the desired anomer with goodselectivity and a high yield. Thus, based on the findings, we haveachieved this invention.

[0025] Specifically, this invention encompasses the followingembodiments.

[0026] (1) A process for selectively preparing either α or β isomer of a1-phosphorylated saccharide derivative monomer comprising the steps ofphosphorolyzing and isomerizing an anomer mixture of a 1-phosphorylatedsaccharide derivative to give α and β isomers of the 1-phosphorylatedsaccharide derivative monomer and selectively crystallizing one of theseisomers to displace the equilibrium between these anomers.

[0027] (2) A process for selectively preparing either α or β isomer of a1-phosphorylated saccharide derivative monomer comprising the steps ofphosphorolyzing and isomerizing an anomer mixture of a 1-phosphorylatedsaccharide derivative represented by formula (I):

[0028] where R¹ and R² independently represents hydrogen, methyl,protected hydroxymethyl or protected carboxyl; R³ represents acyl; R⁴represents a protective group for hydroxy; X represents halogen, alkoxyor alkylthio; W represents oxygen or sulfur; Z represents oxygen, sulfuror optionally substituted carbon; m represents an integer of 1 to 3; nrepresents 0 or 1; p and q represents an integer of 0 to 4; and rrepresents 0 or 1; provided that p, q, r and n meet the conditions ofp+r≦n+1 and q≦2×(n+1)−2×(p+r) when Z is oxygen or sulfur and of p+r≦n+2and q≦2×(n+2)−2×(p+r) when Z is carbon, to give α and β isomers of the1-phosphorylated saccharide derivative monomer and selectivelycrystallizing one of these isomers to displace the equilibrium betweenthese anomers:

[0029] (3) A process for preparing a 1-phosphorylated saccharidederivative monomer represented by formula (3):

[0030] wherein R¹ and R² independently represents hydrogen, methyl,hydroxymethyl or carboxyl; R³ represents hydrogen or acyl; and X, W, Z,n, p, q and r are as defined for formula (1), comprising the steps ofphosphorolyzing and isomerizing an anomer mixture of a 1-phosphorylatedsaccharide derivative represented by formula (1) to give α and β isomersof the 1-phosphorylated saccharide derivative monomer; selectivelycrystallizing one of these isomers to displace the equilibrium betweenthese anomers; and then removing the protective group represented by R⁴.

[0031] (4) A trimer, dimer or monomer of a 1-phosphorylated saccharidederivative represented by formula (4):

[0032] wherein R¹ and R² independently represents hydrogen, methyl,hydroxymethyl protected with substituted benzoyl or protected carboxyl;R⁴ represents hydrogen or a protective group for hydroxy; and R³, X, W,Z, m, n, p, q and r are as defined for formula (1), or salts thereof.

[0033] (5) A 1-phosphorylated saccharide derivative monomer representedby formula (5):

[0034] wherein p and q represents an integer of 0 to 3; r represents 0or 1; and R¹, R², R³, R⁴, X, W and Z are as defined for formula (1);provided that p, q and r meet the conditions of p+q+r≦3 when Z is oxygenor sulfur and of p+q+r≦5 when Z is carbon, or salts thereof.

[0035] (6) A 1-phosphorylated saccharide derivative monomer representedby formula (6):

[0036] wherein R¹ and R² independently represents hydrogen, methyl,hydroxymethyl or carboxy; and R³, X, W, Z, n, p, q and r are as definedfor formula (1), or salts thereof.

[0037] (7) A 1-phosphorylated saccharide derivative monomer representedby formula (7):

[0038] wherein p and q represents an integer of 0 to 3; r represents 0or 1; and R¹, R², R³, R⁴, X, W and Z are as defined for formula (1);provided that p, q and r meet the conditions of p+r≦1, q≦2−2×(p+r) whenZ is oxygen or sulfur and of p+r≦2, q≦4−2×(p+r) when Z is carbon, orsalts thereof.

[0039] (8) A process for preparing a 1-phosphorylated sacchariderepresented by formula (20):

[0040] wherein R¹¹ represents protected hydroxymethyl and R¹⁴ representsa protective group for hydroxy, comprising the steps of treating acompound represented by formula (18):

[0041] wherein R³¹ and R¹⁴ are as defined above, with phosphoric acid inthe presence of a base to give an anomer mixture of a 1-phosphorylatedsaccharide derivative represented by formula (19):

[0042] wherein R¹¹ and R¹⁴ are as defined above and m is as defined inclaim 2; phosphorolyzing and isomerizing the mixture; and displacing theequilibrium between the anomer isomers by selectively crystallizing ana-isomer formed.

[0043] (9) A process for preparing 2-deoxy-α-D-ribose-1-phosphate,comprising the steps of treating a compound represented by formula (18):

[0044] wherein R¹¹ represents protected hydroxymethyl and R¹⁴ representsa protective group for hydroxy, with phosphoric acid in the presence ofa base to give an anomer mixture of a 1-phosphorylated saccharidederivative represented by formula (19):

[0045] wherein R¹¹ and R¹⁴ are as defined above and m is as defined inclaim 2; phosphorolyzing and isomerizing the mixture; displacing theequilibrium between the anomer isomers by selectively crystallizing anα-isomer formed to give the α-isomer; and then removing the protectivegroup.

[0046] We have intensely attempted for achieving the second objectiveand thus have established a highly universal process for preparing anucleoside by utilizing a reverse reaction of nucleoside phosphorylaseswidely distributed in the living world in combination with the abovepreparation processes for a 1-phosphorylated saccharide derivative. Wehave further found that a metal cation capable of forming awater-insoluble salt with a phosphate ion may be present to allow aphosphate ion as a byproduct in the reaction to be precipitated as awater-insoluble salt, resulting in displacement of the reactionequilibrium toward the direction for nucleoside production and thusimprovement in a reaction yield. Thus, we have achieved this inventionproviding a process for preparing a highly pure nucleoside with a lowercost.

[0047] This invention based on the above findings encompasses thefollowing embodiments.

[0048] (10) A process for preparing a nucleoside represented by formula(8):

[0049] wherein B is a base independently selected from the groupconsisting of pyrimidine, purine, azapurine and deazapurine optionallysubstituted by halogen, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl,amino, alkylamino, hydroxy, hydroxyamino, aminoxy, alkoxy, mercapto,alkylmercapto, aryl, aryloxy or cyano; and R¹, R², R³, X, W, Z, n, p, qand r are as defined for formula (1), comprising the first procedure inthe above (3) for preparing a 1-phosphorylated saccharide derivativemonomer comprising the steps of phosphorolyzing and isomerizing ananomer mixture of a 1-phosphorylated saccharide derivative to give α andβ isomers of the 1-phosphorylated saccharide derivative monomer;selectively crystallizing one of these isomers to displace theequilibrium between these anomers; and then removing the protectivegroup represented by R⁴; and

[0050] the second procedure of conducting an exchange reaction of thephosphate group in the 1-phosphorylated saccharide derivative obtainedin the first procedure with a base by the action of a nucleosidephosphorylase.

[0051] (11) A process for preparing a nucleoside represented by formula(9):

[0052] wherein B is as defined for formula (8); and R¹, R², R³, R⁴, X,W, Z, n, p, q and r are as defined for formula (1), comprising anexchange reaction of the phosphate group in the 1-phosphorylatedsaccharide derivative monomer in the above (6) with a base by the actionof a nucleoside phosphorylase.

[0053] (12) A process for preparing a nucleoside represented by formula(10):

[0054] wherein B is as defined for formula (8); and R¹, R², R³, R⁴, X,W, Z, p, q and r are as defined for formula (1), comprising an exchangereaction of the phosphate group in the 1-phosphorylated saccharidederivative monomer in the above (7) with a base by the action of anucleoside phosphorylase.

[0055] (13) A process for preparing a nucleoside represented by formula(21):

[0056] wherein B is as defined for formula (8) in claim 11, comprising

[0057] the first procedure of preparing 2-deoxy-α-D-ribose-1-phosphatein the above (12) where R¹ is hydroxymethyl, R² is hydrogen, p and r are0, and X is fluorine; and

[0058] the second procedure of conducting an exchange reaction of thephosphate group in the 1-phosphorylated saccharide derivative obtainedin the first procedure with a base by the action of a nucleosidephosphorylase.

[0059] In the embodiments of the above (10) to (13), a nucleosidephosphorylase may be at least one selected from the group consisting ofpurine nucleoside phosphorylase (EC2.4.2.1), guanosine nucleosidephosphorylase (EC2.4.2.15), pyrimidine nucleoside phosphorylase(EC2.4.2.2), uridine nucleoside phosphorylase (EC2.4.2.3), thymidinenucleoside phosphorylase (EC2.4.2.4) and deoxyuridine nucleosidephosphorylase (EC2.4.2.23).

[0060] A nucleoside phosphorylase activity may be obtained using amicroorganism expressing at least one nucleoside phosphorylase selectedfrom the group consisting of purine nucleoside phosphorylase(EC2.4.2.1), guanosine nucleoside phosphorylase (EC2.4.2.15), pyrimidinenucleoside phosphorylase (EC2.4.2.2), uridine nucleoside phosphorylase(EC2.4.2.3), thymidine nucleoside phosphorylase (EC2.4.2.4) anddeoxyuridine nucleoside phosphorylase (EC2.4.2.23).

[0061] In the embodiments of the above (10) to (13), a metal cationcapable of forming a water-insoluble salt with a phosphate ion may bepresent in the reaction solution during the exchange reaction of aphosphate group in the 1-phosphorylated saccharide derivative monomerwith a base by the action of a nucleoside phosphorylase.

[0062] The metal cation capable of forming a water-insoluble salt withthe phosphate ion in the embodiments of the above (10) to (13) may be atleast one metal cation selected from the group consisting of calcium,barium, aluminum and magnesium ions.

[0063] Furthermore, this invention encompasses a compound represented byany of formulas (11) to (13) and (20).

[0064] That is, this invention also encompasses:

[0065] a synthetic nucleoside, which is not naturally producedrepresented by formula (11):

[0066] wherein B, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are as definedfor formulas (1) and (8) or its salt, excluding trifluorothymidine,ribavirin, orotidine, uracil arabinoside, adenine arabinoside,2-methyl-adenine arabinoside, 2-chloro-hypoxanthine arabinoside,thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosinearabinoside, guanine arabinoside, thymine arabinoside, enocitabine,gemcitabine, azidothymidine, idoxuridine, dideoxyadenosine,dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine,thiadideoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine,tegafur, doxifluridine, bredinin, nebularine, allopurinol uracil,5-fluorouracil, 2′-aminouridine, 2′-aminoadenosine, 2′-aminoguanidine,2-chloro-2′-aminoinosine, DMDC and FMDC;

[0067] a synthetic nucleoside, which is not naturally producedrepresented by formula (12):

[0068] wherein B, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are as definedfor formulas (1) and (8) or its salt, excluding trifluorothymidine,ribavirin, orotidine, uracil arabinoside, adenine arabinoside,2-methyl-adenine arabinoside, 2-chloro-hypoxanthine arabinoside,thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosinearabinoside, guanine arabinoside, thymine arabinoside, enocitabine,gemcitabine, azidothymidine, idoxuridine, dideoxyadenosine,dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine,thiadideoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine,tegafur, doxifluridine, bredinin, nebularine, allopurinol uracil,5-fluorouracil, 2′-aminouridine, 2′-aminoadenosine, 2′-aminoguanidine,2-chloro-2′-aminoinosine, DMDC and FMDC;

[0069] a nucleoside represented by formula (13):

[0070] wherein B, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are as definedfor formulas (1) and (8) or its salt, excluding trifluorothymidine,ribavirin, orotidine, uracil arabinoside, adenine arabinoside,2-methyl-adenine arabinoside, 2-chloro-hypoxanthine arabinoside,thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosinearabinoside, guanine arabinoside, thymine arabinoside, enocitabine,gemcitabine, azidothymidine, idoxuridine, dideoxyadenosine,dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine,thiadideoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine,tegafur, doxifluridine, bredinin, nebularine, allopurinol uracil,5-fluorouracil, 2′-aminouridine, 2′-aminoadenosine, 2′-aminoguanidine,2-chloro-2′-aminoinosine, DMDC and FMDC; and

[0071] a 1-phosphorylated saccharide represented by formula (20):

[0072] wherein R¹¹ and R¹⁴ are as defined for formula (18), or its salt.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0073] This invention will be described in detail.

[0074] Saccharides which may used in this invention include, but notlimited to, residues derived from D- and L-type natural monosaccharidesincluding 6-deoxysaccharides such as fucose, rhamnose, digitoxose,oleandrose and quinovose, hexoses such as allose, altrose, glucose,mannose, gulose, idose, galactose and talose, pentoses such as ribose,arabinose, xylose and lyxose, tetroses such as erythrose and threose,aminosaccharides such as glucosamine and daunosamine, uronic acids suchas glucuronic acid and galacturonic acid, ketoses such as psicose,fructose, sorbose, tagatose and pentulose, and deoxysaccharides such as2-deoxyribose; residues derived from synthetic pyranose and furanosesaccharides; and saccharide residue derivatives in which hydroxy and/oramino groups in any of the above residues are protected or acylated orsaccharides having a halogenated saccharide residue in which hydroxy isreplaced with halogen such as fluorine.

[0075] In this invention, a 1-phosphorylated saccharide derivativerefers to a saccharide derivative in which among residues derived fromnatural or synthetic monosaccharide, 1-hydroxy is phosphorylated. Unlessotherwise indicated, it may include a monomer, dimer or trimer or amixture thereof, where there are no restrictions to its mixture ratio.

[0076] A protective group in terms of “protected hydroxymethyl” and “aprotective group of hydroxy” means that which may be removed by anappropriate chemical process such as hydrogenolysis, hydrolysis andphotolysis, including formyl, acyl, silyl, alkyl, aralkyl, carbonyl,preferably formyl, aliphatic acyl, aromatic acyl, silyl, alkoxyalkyl,halogenated alkyl, aralkyl, alkoxycarbonyl and aralkyloxycarbonyl.

[0077] Aliphatic acyl may be alkylcarbonyl and halogenated loweralkylcarbonyl.

[0078] Examples of alkylcarbonyl include acetyl, propionyl, butyryl,isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl,nonylcarbonyl, decylcarbonyl, 3-methylnonylcarbonyl,8-methylnonylcarbonyl, 3-ethyloctylcarbonyl, 3,7-dimethyloctylcarbonyl,undecylcarbonyl, dodecylcarbonyl, tridecylcarbonyl, tetradecylcarbonyl,pentadecylcarbonyl, hexadecylcarbonyl, 1-methylpentadecylcarbonyl,14-methylpentadecylcarbonyl, 13,13-dimethyltetradecylcarbonyl,heptadecylcarbonyl, 15-methylhexadecylcarbonyl and octadecylcarbonyl.

[0079] Examples of halogenated lower alkylcarbonyl include chloroacetyl,dichloroacetyl, trichloroacetyl and trifluoroacetyl.

[0080] Aromatic acyl may be arylcarbonyl, halogenated arylcarbonyl,lower-alkylated arylcarbonyl, lower-alkoxylated arylcarbonyl, nitratedarylcarbonyl, lower-alkoxycarbonylated arylcarbonyl or arylatedarylcarbonyl.

[0081] Examples of arylcarbonyl include benzoyl, α-naphthoyl andβ-naphthoyl.

[0082] Examples of halogenated arylcarbonyl include 2-fluorobenzoyl,3-fluorobenzoyl, 4-fluorobenzoyl, 2-chlorobenzoyl, 3-chlorobenzoyl,4-chlorobenzoyl, 2-bromobenzoyl, 3-bromobenzoyl, 4-bromobenzoyl,2,4-dichlorobenzoyl, 2,6-dichlorobenzoyl, 3,4-dichlorobenzoyl and3,5-dichlorobenzoyl.

[0083] Examples of lower-alkylated arylcarbonyl include 2-toluoyl,3-toluoyl, 4-toluoyl and 2,4,6-trimethylbenzoyl.

[0084] Examples of lower-alkoxy arylcarbonyl include 2-anisoyl,3-anisoyl and 4-anisoyl.

[0085] Examples of nitrated arylcarbonyl include 2-nitrobenzoyl,3-nitrobenzoyl, 4-nitrobenzoyl and 3,5-dinitrobenzoyl.

[0086] Examples of lower-alkoxycarbonylated arylcarbonyl include2-(methoxycarbonyl)benzoyl. Examples of arylated arylcarbonyl include4-phenylbenzoyl.

[0087] Silyl may be lower-alkylsilyl and aryl-substitutedlower-alkylsilyl.

[0088] Examples of lower-alkyl silyl include trimethylsilyl,triethylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl,methyldiisopropylsilyl and triisopropylsilyl.

[0089] Examples of aryl-substituted lower-alkylsilyl includediphenylmethylsilyl, diphenylisopropylsilyl and phenyldiisopropylsilyl.

[0090] Aralkyl may be benzyl, aralkyl substituted with lower alkyl,aralkyl substituted with lower alkoxy, aralkyl substituted with nitro,aralkyl substituted with halogen or aralkyl substituted with cyano.

[0091] Examples of these include 2-methylbenzyl, 3-methylbenzyl,4-methylbenzyl, 2,4,6-trimethylbenzyl, 2-methoxybenzyl, 3-methoxybenzyl,4-methoxybenzyl, 2-nitrobenzyl, 3-nitrobenzyl, 4-nitrobenzyl,2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-bromobenzyl,3-bromobenzyl, 4-bromobenzyl, 2-cyanobenzyl, 3-cyanobenzyl and4-cyanobenzyl.

[0092] Aralkyloxycarbonyl may be aralkyloxycarbonyl substituted withlower alkyl, aralkyloxycarbonyl substituted with lower alkoxy,aralkyloxycarbonyl substituted with nitro, aralkyloxycarbonylsubstituted with halogen or aralkyloxycarbonyl substituted with cyano.

[0093] Examples of these include 2-methylbenzyloxycarbonyl,3-methylbenzyloxycarbonyl, 4-methylbenzyloxycarbonyl,2,4,6-trimethylbenzyloxycarbonyl, 2-methoxybenzyloxycarbonyl,3-methoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, 3-nitrobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,3-chlorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl,2-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 2-cyanobenzyloxycarbonyl,3-cyanobenzyloxycarbonyl and 4-cyanobenzyloxycarbonyl.

[0094] Alkoxycarbonyl may be lower-alkoxycarbonyl, alkoxycarbonylsubstituted with halogen or alkoxycarbonyl substituted with alkylsilyl.

[0095] Examples of lower-alkoxycarbonyl include methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, sec-butoxycarbonyl andtert-butoxycarbonyl.

[0096] Examples of alkoxycarbonyl substituted with halogen include2,2,2-trichloroethoxycarbonyl. Examples of alkoxycarbonyl substitutedwith alkylsilyl include 2-trimethylsilylethoxycarbonyl.

[0097] Alkyl may be alkoxyalkyl such as methoxyethyl, ethoxymethyl,2-methoxyethyl and 2-methoxyethoxymethyl; halogenated alkyl such as2,2,2-trichloroethyl; or lower alkyl substituted with aryl such asbenzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl andtriphenylmethyl.

[0098] Among these, aliphatic acyl, aromatic acyl and aralkyl arepreferable; 4-toluoyl, 4-chlorobenzoyl and benzyl are more preferable. Aprotective group in terms of “protected carboxyl” in R¹ and R² refers tothat which may be removed by an appropriate chemical process such ashydrogenolysis, hydrolysis and photolysis, including preferably loweralkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl and tert-butyl; silylated lower alkyl such as2-(trimethylsilyl)ethyl and 2-(triethylsilyl)ethyl; or the above aralkylor alkoxyalkyl, more preferably methyl, tert-butyl or benzyl.

[0099] Halogen in terms of X refers to fluorine, chlorine, bromine oriodine.

[0100] Alkoxy and alkylthio in terms of X may be alkoxy and alkylthiohaving the above lower alkyl, aralkyl or alkoxyalkyl, preferablymethoxy, methoxyethoxy or methylthio.

[0101] Optionally substituted carbon in terms of Z refers to carbonhaving one or two of the substituent represented by the formula (Xq andNHR³) or when having no substituents, carbon having hydrogen atoms.

[0102] Acyl in terms of R³ may be the above aliphatic acyl, aromaticacyl, alkoxycarbonyl or aralkyloxycarbonyl, or lower-alkanesulfonyl suchas methanesulfonyl and trifluoromethanesulfonyl or arylsulfonyl such asbenzenesulfonyl and p-toluenesulfonyl; preferably, aliphatic acyl,aromatic acyl or lower-alkanesulfonyl; specifically, acetyl,trifluoroacetyl, benzoyl and methanesulfonyl. When more than one of NHR³are used as a substituent, R³s in individual NHR³ independentlyrepresent any of the above radicals.

[0103] A protective group in “protected hydroxymethyl” and “a protectivegroup for hydroxy” in terms of R⁴, R¹¹ and R¹⁴ may be selected fromthose described for R¹ and R².

[0104] Saccharide residues having a structure represented by any offormulas (1) to (17) may be preferably, but not limited to, thosederived from a natural monosaccharide described above, those derivedfrom a synthetic saccharide, derivatives from the saccharide residues orhalogenated saccharide residues, as described above.

[0105] Salts of a compound represented by any of formulas (4) to (7) maybe those formed by a phosphate radical in the compound. Examples of sucha salt include alkali metal salt such as sodium, potassium and lithiumsalts; alkaline earth metal salt such as magnesium, calcium and bariumsalts; metal salt such as aluminum and iron salts; ammonium salt; oralkylamine salt such as primary, secondary and tertiary alkyl aminesalts.

[0106] Primary amine herein may be alkylamine such as methylamine,ethylamine, propylamine, isopropylamine, butylamine, hexylamine andoctylamine; cycloalkylamine such as cyclohexylamine; or benzylamine.

[0107] Secondary amine may be dialkylamine such as diethylamine,diisopropylamine, dibutylamine, dihexylamine and dioctylamine;dicycloalkylamine such as dicyclohexylamine; or cyclic amine such aspiperidine, morpholine and N-methylpiperadine.

[0108] Tertiary amine may be tertiary-alkylamine such as trimethylamine,triethylamine, tripropylamine, N-ethyldiisopropylamine, tributylamine,trihexylamine, trioctylamine, N-ethyldicyclohexylamine,N-methylpiperidine, N-methylmorpholine andN,N,N′,N′-tetramethylethylenediamine; aniline compound such as aniline,N,N-dimethylaniline, N,N-diethylaniline, N,N-dibutylaniline andN,N-dioctylaniline; pyridine compound such as pyridine,2,6-dimethylpyridine, 2,4,6-lutidine and nicotinamide; amino acid suchas glycine, alanine, proline, lysine, arginine and glutamine; oroptically active amine such as cinchonidine, 1-(1-naphthyl)ethylamineand 1-phenylethylamine, all of which include monovalent and bivalentsalts.

[0109] A compound represented by any of formula (4) to (7) of thisinvention may absorb moisture to have adsorbed water or become ahydrate, all of which may be encompassed by this invention.

[0110] An anomer mixture of a 1-phosphorylated saccharide derivativeaccording to this invention may be prepared by, but not limited to, areaction represented by reaction formula (I):

[0111] Reaction Formula (I)

[0112] In this formula, R¹, R², R³, R⁴, X, W, Z, m, n, p, q and r are asdefined for formula (1), and Y represents fluorine, chlorine, bromine oriodine. When m is 1, 2 or 3, phosphoric acid tri-, di- or mono-ester isprovided, respectively. These are referred to as 1-phosphorylatedsaccharide derivative trimer, 1-phosphorylated saccharide derivativedimer and 1-phosphorylated saccharide derivative monomer, respectively.Furthermore, 1-phosphorylated saccharide derivative trimer,1-phosphorylated saccharide derivative dimer and 1-phosphorylatedsaccharide derivative monomer are collectively referred to as a1-phosphorylated saccharide derivative, for which there are norestrictions to its mixture ratio.

[0113] A preferable phosphoric acid may be, but not limited to, one witha lower water content such as orthophosphoric acid.

[0114] There are no restrictions to a base as long as it does notinhibit the reaction and functions as a deoxidizer. Preferable inorganicbases include carbonates and hydroxides of alkali and alkaline earthmetals. Preferable organic bases include tertiary alkylamines, anilines,pyridines and optically active amines.

[0115] A dehydrating agent may be used when moisture from a solvent oran additive adversely affects the reaction. There are no restrictions toa dehydrating agent as long as it has adequate adsorptivity orreactivity with water; preferably molecular sieves and phosphoruspentoxide.

[0116] The reaction is generally conducted in the presence of a solvent.There are no restrictions to a solvent as long as it does not inhibitthe reaction and dissolve starting materials to some degree. Solventswhich may be used include aliphatic hydrocarbons such as hexane andheptane; aromatic hydrocarbons such as benzene, toluene, xylene andanisole; halogenated hydrocarbons such as methylene chloride,chloroform, carbon tetrachloride, dichloroethane, chlorobenzene anddichlorobenzene; esters such as ethyl formate, ethyl acetate, propylacetate, n-butyl acetate and diethyl carbonate; ethers such as diethylether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane anddiglyme; nitriles such as acetonitrile, propionitrile andisobutylnitrile; amides such as formamide, N,N-dimethylformamide,N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone andN,N-dimethyl-2-imidazolydinone; ketones such as acetone, 2-butanone,methyl isopropyl ketone and methyl isobutyl ketone; and a mixture of twoor more selected therefrom.

[0117] There are no restrictions to a reaction temperature; generally-80° C. to 60° C., preferably −10° C. to 25° C.

[0118] A reaction period may vary depending on many factors such asstarting materials, reagents, the type of a solvent and a reactiontemperature; generally 1 min to 24 hours, preferably 10 min to 2 hoursfor completing the reaction.

[0119] There are no restrictions. to a ratio of a saccharide derivative(14) to phosphoric acid; the reaction is generally conducted with aratio of compound (14): phosphoric acid=1:10 to 3:1. In this case, theproduct (1) may be a mixture of the compounds whose saccharide residuenumber (i.e., m) coupled with phosphoric acid is 1, 2 or 3, depending onthe ratio of compound (14): phosphoric acid.

[0120] Furthermore, a 1-phosphorylated saccharide derivative (16a) or(16b) with either α- or β-form may be prepared by a reaction representedby reaction formula (II):

[0121] In this formula, R¹, R², R³, R⁴, X, W, Z, m, n, p, q and r are asdefined for formula (1).

[0122] According to this preparation process, the 1-phosphorylatedsaccharide derivative represented by formula (15) may be a monomer,dimer or trimer or a mixture thereof in any mixture ratio because theymay be converted into the 1-phosphorylated saccharide derivativerepresented by formula (16) in the reaction system.

[0123] A preferable phosphoric acid may be, but not limited to, one witha lower water content such as orthophosphoric acid.

[0124] A base is important for forming a salt with the phosphate groupin compound (16) to selectively crystallize one of α- and β-compounds,(16a) or (16b). The most suitable base may be selected in the light of asolvent used in the reaction; preferably, the above inorganic bases,tertiary alkylamines, anilines, pyridines, amino acids and opticallyactive amines, and salts formed include monovalent and bivalent salts.

[0125] A dehydrating agent may be used when moisture from a solvent oran additive adversely affects the reaction. There are no restrictions toa dehydrating agent as long as it has adequate adsorptivity orreactivity with water; preferably molecular sieves and phosphoruspentoxide.

[0126] The reaction is generally conducted in the presence of a solvent.There are no restrictions to a solvent as long as it does not inhibitthe reaction, dissolve starting materials to some degree and promotesselective crystallization of one of the α- and β-forms, (16a) or (16b),generated by salt formation of the phosphate group in compound (16),including the above aliphatic hydrocarbons, aromatic hydrocarbons,halogenated hydrocarbons, esters, ethers, nitrites, amides, ketones anda mixture of two or more selected therefrom.

[0127] There are no restrictions to a reaction temperature as long as itaccelerates the equilibrium reaction between compounds (15) and (16) forpromoting selective crystallization of one of the α- and β-forms, (16a)or (16b), generated by salt formation of the phosphate group in compound(16); generally −80° C. to 60° C., preferably −10° C. to 25° C.

[0128] A reaction period may vary depending on many factors such asstarting materials, reagents, the type of a solvent and a reactiontemperature; generally 3 hours to 1 week, preferably 6 hours to 24 hoursfor completing the reaction.

[0129] There are no restrictions to a ratio of a saccharide derivative(1) to phosphoric acid; the reaction is generally conducted with a ratioof compound (1): phosphoric acid=1:10 to 3:1, where the pH of thereaction system is generally 1 to 7, suitably in an acidic range from 1to 4.

[0130] The 1-phosphorylated saccharide derivative (16a) or (16b) witheither α- or β-form may be isolated as a phosphate with a base otherthan that used in the reaction system by a salt-exchange reaction.

[0131] Bases which may be herein used include the above inorganic bases,primary alkylamines, secondary alkylamines, tertiary alkylamines,anilines, pyridines, amino acids and optically active amines, and saltsformed include monovalent and bivalent salts.

[0132] The protective group may be removed by reaction formula (III) toprepare a 1-phosphorylated saccharide derivative (17a) or (17b).

[0133] Reaction Formula (III)

[0134] In this formula, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are asdefined for formula (1); R¹ and R² independently represent hydrogen,methyl, hydroxymethyl or carboxyl; and R³ represents hydrogen or acyl.

[0135] When using the above aliphatic acyl, aromatic acyl or alkoxycarbonyl as a protective group for hydroxymethyl in R¹ and R² or hydroxyin R⁴, or using the above lower alkyl as a protective group for carboxylin R¹ and R² in compound (16a) or (16b), it may be removed by treatingthe compound with a base in an aqueous solvent. Bases which may be usedinclude preferably alkali metal carbonates such as sodium carbonate andpotassium carbonate; alkali metal hydroxides such as lithium hydroxide,sodium hydroxide and potassium hydroxide; ammonium hydroxides such asammonium hydroxide and tetra-n-butylammonium hydroxide; and the aboveinorganic bases, primary alkylamines, secondary alkylamines and tertiaryalkylamines.

[0136] Solvents which may be used include, with no restrictions, thoseused in a common hydrolysis; preferably water; alcohols such asmethanol, ethanol, n-propanol and isopropanol; and the above ethers. Areaction temperature and a reaction period vary, with no restrictions,depending on many factors such as starting materials and a base used;generally the reaction may be completed at −10° C. to 100° C. for 1 hourto 5 days. The protective group R³ may be left or simultaneously removedas appropriate by adjusting a reaction temperature, a reaction periodand the equivalent values of reagents.

[0137] When using the above aralkyl or aralkyloxycarbonyl as aprotective group for hydroxymethyl in R¹ and R² or hydroxy in R⁴ orusing the above aralkyl as a protective group for carboxy in R¹ and R²in compound (16a) or (16b), they may be removed by, for example,catalytic hydrogenation using a metal catalyst.

[0138] The catalyst may be preferably selected from palladium-carbon,Raney nickel, platinum oxide, platinum black, rhodium-aluminum oxide,triphenylphosphine-rhodium chloride and palladium-barium sulfate. Thereare no restrictions to a reaction pressure. Generally, a solvent usedmay be any of those used in a common hydrolysis with no restrictions. Itmay be preferably selected from water; alcohols such as methanol,ethanol, n-propanol and isopropanol; the above ethers; and the aboveesters. A reaction temperature and a reaction period vary, with norestrictions, depending on many factors such as starting materials and abase used; generally the reaction may be completed at −10° C. to 100° C.for 1 hour to 5 days. The protective group R³ may be generally left.

[0139] When using the above silyl as a protective group forhydroxymethyl in R¹ and R² or hydroxy in R⁴ or using the above silylatedlower alkyl as a protective group for carboxy in R¹ and R² in compound(16a) or (16b), they may be removed by, for example, using a compoundwhich can generate fluoride anion such as tetra-n-butylammoniumfluoride.

[0140] There are no restrictions to a solvent as long as it does notinhibit the reaction; for example, the above ethers may be used. Areaction temperature and a reaction period vary, with no restrictions,depending on many factors such as starting materials and a base used;generally the reaction may be completed at −10° C. to 50° C. for 10 minto 10 hours. The protective group R³ may be generally left.

[0141] In removing any protective group, a phosphate group in a productis obtained as a salt with a base present in a reaction system. The saltmay be, if necessary, converted into a salt with another base. In such acase, a base used may be selected from the above inorganic bases,primary alkylamines, secondary alkylamines, tertiary alkylamines,anilines, pyridines, amino acids and optically active amines, and saltsformed include monovalent and bivalent salts.

[0142] A 1-phosphorylated saccharide derivative as used herein is asaccharide or its derivative in which a phosphoric acid moiety iscoupled at 1-position via an ester linkage.

[0143] It may be specifically represented by formula (6):

[0144] wherein R¹ and R² independently represent hydrogen, methyl,hydroxymethyl or carboxy; R³, X, W, Z, n, p, q and r are as defined forformula (4).

[0145] Typical examples include, but not limited to, ribose-1-phosphate,2-deoxyribose-1-phosphate, 2,3-dideoxyribose-1-phosphate andarabinose-1-phosphate, but any derivative may be used withoutdistinction as long as it can be obtained by any of the above highlyuniversal and anomer-selective preparation processes.

[0146] Examples of a saccharide derived from a natural product whichconstitutes a 1-phosphorylated saccharide derivative include, but notlimited to, aldopentoses such as D-arabinose, L-arabinose, D-xylose,L-lyxose and D-ribose; ketopentoses such as D-xylose, L-xylose andD-ribulose; aldohexoses such as D-galactose, L-galactose, D-glucose,D-talose and D-mannose; ketohexoses such as D-tagatose, L-sorbose,D-psicose and D-fructose; deoxysaccharides such as D-2-deoxyribose,D-2,3,-dideoxyribose, D-fucose, L-fucose, D-rhamnose, L-rhamnose,D-fucopyranose, L-fucopyranose, D-rhamnofuranose, L-rhamnofuranose,D-allomethylose, D-quinovose, D-antiallose, D-talomethylose,L-talomethylose, D-digitalose, D-digitoxose, D-cymarose, tyvelose,abequose, paratose, colitose and ascarilose; aminosaccharides such asglucosamine and daunosamine; and uronic acids such as glucuronic acidand galacturonic acid.

[0147] There will be described a process for preparing a nucleosideaccording to this invention. A base used in this process is a natural orsynthetic base selected from pyrimidine, purine, azapurine anddeazapurine, which may be substituted with halogen, alkyl, haloalkyl,alkenyl, haloalkenyl, alkynyl, amino, alkylamino, hydroxy, hydroxyamino,aminoxy, alkoxy, mercapto, alkylmercapto, aryl, aryloxy and/or cyano.

[0148] Examples of halogen as a substituent include chlorine, fluorine,bromine and iodine. Examples of alkyl include lower alkyls with 1 to 7carbon atoms such as methyl, ethyl and propyl. Examples of haloalkylinclude those having an alkyl with 1 to 7 carbon atoms such asfluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl andbromoethyl. Examples of alkenyl include those with 2 to 7 carbon atomssuch as vinyl and allyl. Examples of haloalkyl include those havingalkenyl with 2 to 7 carbon atoms such as bromovinyl and chlorovinyl.Examples of alkynyl include those with 2 to 7 carbon atoms such asethynyl and propynyl. Examples of alkylamino include those having alkylwith 1 to 7 carbon atoms such as methylamino and ethylamino. Examples ofalkoxy include those with 1 to 7 carbon atoms such as methoxy andethoxy. Examples of alkylmercapto include those having alkyl with 1 to 7carbon atoms such as methylmercapto and ethylmercapto. Examples of arylinclude phenyl; alkylphenyls having alkyl with 1 to 5 carbon atoms suchas methylphenyl and ethylphenyl; alkoxyphenyls having alkoxy with 1 to 5carbon atoms such as methoxyphenyl and ethoxyphenyl; alkylaminophenylshaving alkylamino with 1 to 5 carbon atoms such as dimethylaminophenyland diethylaminophenyl; and halogenophenyls such as chlorophenyl andbromophenyl.

[0149] Examples of a pyrimidine base include cytosine, uracil,5-fluorocytosine, 5-fluoro uracil, 5-chlorocytosine, 5-chlorouracil,5-bromocytosine, 5-bromouracil, 5-iodocytosine, 5-iodouracil,5-methylcytosine, 5-methyluracil (thymine), 5-ethylcytosine,5-ethyluracil, 5-fluoromethylcytosine, 5-fluoromethyluracil,5-trifluorocytosine, 5-trifluorouracil, 5-vinyluracil,5-bromovinyluracil, 5-chlorovinyluracil, 5-ethynylcytosine,5-ethynyluracil, 5-propynyluracil, pyrimidin-2-one,4-hydroxyaminopyrimidin-2-one, 4-aminoxypyrimidin-2-one,

[0150] 4-methoxypyrimidin-2-one, 4-acetoxypyrimidin-2-one,4-fluoropyrimidin-2-one and 5-fluoropyrimidin-2-one.

[0151] Examples of a purine base include purine, 6-aminopurine(adenine), 6-hydroxypurine, 6-fluoropurine, 6-chloropurine,6-methylaminopurine, 6-dimethylaminopurine,6-trifluoromethylaminopurine, 6-benzoylaminopurine, 6-acetylaminopurine,6-hydroxyaminopurine, 6-aminoxypurine, 6-methoxypurine, 6-acetoxypurine,6-benzoyloxypurine, 6-methylpurine, 6-ethylpurine,6-trifluoromethylpurine, 6-phenylpurine, 6-mercaptopurine,6-methylmercaptopurine, 6-aminopurine-1-oxide, 6-hydroxypurine-1-oxide,2-amino-6-hydroxypurine (guanine), 2,6-diaminopurine,2-amino-6-chloropurine, 2-amino-6-iodopurine, 2-aminopurine,2-amino-6-mercaptopurine, 2-amino-6-methylmercaptopurine,2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine,2-amino-6-benzoyloxypurine, 2-amino-6-acetoxypurine,2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine,2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine,6-amino-2-chloropurine (2-chloroadenine) and 6-amino-2-fluoropurine(2-fluoroadenine).

[0152] Examples of an azapurine and a deazapurine bases include6-amino-3-deazapurine, 6-amino-8-azapurine,2-amino-6-hydroxy-8-azapurine, 6-amino-7-deazapurine,6-amino-1-deazapurine and 6-amino-2-azapurine.

[0153] A nucleoside phosphorylase is a generic name for enzymes capableof phosphorolysis of an N-glycoside bond in a nucleoside in the presenceof phosphoric acid and this invention utilizes its reverse reaction. Anenzyme used in the reaction may be of any type or origin as long as ithas an activity of forming a desired nucleoside from a corresponding1-phosphorylated saccharide derivative and a base. The enzymes may begenerally categorized into two types, purine and pyrimidine types.Examples of a purine type enzyme include purine nucleoside phosphorylase(EC2.4.2.1) and guanosine nucleoside phosphorylase (EC2.4.2.15).Examples of a pyrimidine type enzyme include pyrimidine nucleosidephosphorylase (EC2.4.2.2), uridine nucleoside phosphorylase (EC2.4.2.3),thymidine nucleoside phosphorylase (EC2.4.2.4) and deoxyuridinenucleoside phosphorylase (EC2.4.2.23).

[0154] A microorganism expressing a nucleoside phosphorylase in thisinvention may be, with no restrictions, any microorganism expressing atleast one nucleoside phosphorylase selected from the group consisting ofpurine nucleoside phosphorylase (EC2.4.2.1), guanosine nucleosidephosphorylase (EC2.4.2.15), pyrimidine nucleoside phosphorylase(EC2.4.2.2), uridine nucleoside phosphorylase (EC2.4.2.3), thymidinenucleoside phosphorylase (EC2.4.2.4) and deoxyuridine nucleosidephosphorylase (EC2.4.2.23).

[0155] Preferable examples of such a microorganism include strainsbelonging to Nocardia, Microbacterium, Corynebacterium, Brevibacterium,Cellulomonas, Flabobacterium, Kluyvere, Micobacterium, Haemophilus,Micoplana, Protaminobacter, Candida, Saccharomyces, Bacillus,thermophile Bacillus, Pseudomonas, Micrococcus, Hafnia, Proteus, Vibrio,Staphyrococcus, Propionibacterium, Sartina, Planococcus, Escherichia,Kurthia, Rhodococcus, Acinetobacter, Xanthobacter, Streptomyces,Rhizobium, Salmonella, Klebsiella, Enterobacter, Erwinia, Aeromonas,Citrobacter, Achromobacter, Agrobacterium, Arthrobacter andPseudonocardia.

[0156] Recent advance in molecular biology and genetic engineering hasallowed us to analyze molecular-biological properties, an amino acidsequence and so-on of a nucleoside phosphorylase in the above strain forobtaining the gene for the protein from the strain, to constitute arecombinant plasmid in which a control region required for the gene andits expression is inserted, to introduce the plasmid into a given hostand to produce a gene recombinant strain expressing the protein, andthese processes have become relatively easier. In the light of therecent technical level, such a gene recombinant strain in which a genefor a nucleoside phosphorylase is introduced in a given host shall bealso included in a microorganism expressing a nucleoside phosphorylaseaccording to this invention.

[0157] A control region required for expression herein may be a promotersequence (including an operator sequence controlling transcription), aribosome binding sequence (SD sequence), a transcription terminationsequence, or the like. Examples of a promoter sequence include a trpoperator in a tryptophane operon derived from E. coli; a lac promoter ina lactose operon; a PL and a PR promoters derived from lambda phage; agluconate synthase promoter (gnt) derived from Bacillus subtilis; analkali protease promoter (apr); a neutral protease promoter (npr); andα-amylase promoter (amy). A uniquely modified and designed sequence suchas a tac promoter may be used. A ribosome linkage sequence may be, forexample, a sequence derived from E. coli or Bacillus subtilis, but anysequence may be used as long as it can function in a desired host suchas E. coli and Bacillus subtilis. For example, one can use a consensussequence formed by DNA synthesis, that is, a sequence with more than 4consecutive bases complementary to 3′-terminal region in 16S ribosomeRNA. A transcription termination sequence is not always necessary, but aρ-factor independent terminator such as a lipoprotein terminator and atrp operon terminator may be used. Desirably, these control regions on arecombinant plasmid may be sequentially aligned as follows; fromupstream of 5′-terminal, a promoter sequence, a ribosome linkagesequence, a nucleoside phosphorylase coding gene and a transcriptiontermination sequence.

[0158] As examples of a plasmid herein, pBR 322, pUC18, Bluescript IISK(+), pKK223-3 and pSC101 having an autonomously replicable region inE. coli; pUB110, pTZ4, pC194, ρ11, φ1 and φ105 having an autonomouslyreplicable region in Bacillus subtilis may be used as a vector. Asexamples of a plasmid autonomously replicable in two or more hosts,pHV14, TRp7, Yep7 and pBS7 may be used as a vector.

[0159] A given host herein may be typically, but not limited to,Escherichia coli as described in Examples later, but other strains suchas Bacillus sp. including Bacillus subtilis, yeasts and actinomyces maybe used.

[0160] Nucleoside phosphorylase activity in this invention may beobtained from, besides the above strains having the enzyme activity, aprocessed material of the strain exhibiting the enzyme activity and animmobilized product thereof. A processed material of the strain may be,for example, acetone-dried strain or a bacterial debris prepared by anappropriate procedure such as mechanical destruction, ultrasonicdisintegration, freezing and thawing, pressurization anddepressurization, osmotic pressure method, autolysis, cell-walldecomposition and surfactant treatment. If necessary, the strain may befurther purified by ammonium sulfate precipitation, acetoneprecipitation or column chromatography.

[0161] In this invention, a metal cation capable of forming awater-insoluble salt with phosphate ion may be, without restriction, anymetal cation which can form a water-insoluble salt with phosphate ion asa byproduct in the reaction and may be precipitated; for example,calcium, magnesium, barium, iron, cobalt, nickel, copper, silver,molybdenum, lead, zinc and lithium ions. Among these, particularlypreferable are industrially universal and safe metal ions which do notadversely affect the reaction, e.g., calcium, barium, aluminum andmagnesium ions.

[0162] A metal cation capable of forming a water-insoluble salt withphosphate ion in this invention may be obtained by adding a salt of ametal cation capable of forming a water-insoluble salt with phosphateion with at least one anion selected from chloride, nitride, carbonate,sulfate, acetate and hydroxyl ions into the reaction solution. Examplesof such a salt include calcium chloride, calcium nitride, calciumcarbonate, calcium sulfate, calcium acetate, barium chloride, bariumnitride, barium carbonate, barium sulfate, barium acetate, aluminumchloride, aluminum nitride, aluminum carbonate, aluminum sulfate,aluminum acetate, calcium hydroxide, barium hydroxide, aluminumhydroxide, magnesium hydroxide, magnesium chloride, magnesium nitride,magnesium carbonate, magnesium sulfate and magnesium acetate.

[0163] Such a metal cation may be present as a salt with apentose-1-phosphate in the reaction solution; for example,ribose-1-phosphate calcium salt, 2-deoxyribose-1-phosphate calcium salt,2,3-dideoxyribose-1-phosphate calcium salt, arabinose-1-phosphatecalcium salt, ribose-1-phosphate barium salt, 2-deoxyribose-1-phosphatebarium salt, 2,3-dideoxyribose-1-phosphate barium salt,arabinose-1-phosphate barium salt, ribose-1-phosphate aluminum salt,2-deoxyribose-1-phosphate aluminum salt, 2,3-dideoxyribose-1-phosphatealuminum -salt and arabinose-1-phosphate aluminum salt.

[0164] A reaction for preparing a nucleoside compound in this inventionmay be conducted under the conditions such as appropriate pH andtemperature and within the control ranges thereof, depending on a targetnucleoside, a 1-phosphorylated saccharide derivative and a base assubstrates, a nucleoside phosphorylase or a microorganism exhibiting theactivity of the enzyme as a reaction catalyst, and the type and theproperties of a metal salt added for removing phosphoric acid from thereaction system; generally at pH 5 to 10 and a temperature of 10 to 60°C. If pH is not within the control range, a reaction inversion rate maybe reduced due to, for example, poor stability of a target product orsubstrate, reduction in enzyme activity and failure to forming awater-insoluble salt with phosphoric acid. If pH varies in the course ofthe reaction, an acid such as hydrochloric acid and sulfuric acid or analkali such as sodium hydroxide and potassium hydroxide may be, whennecessary, added at an appropriate timing. The concentrations of a1-phosphorylated saccharide derivative and a base are suitably about 0.1to 1000 mM. In terms of a molar ratio between them, a molar ratio of abase to a 1-phosphorylated saccharide derivative or its salt may be 1 to10, preferably 0.95 or less in the light of a reaction inversion rate.

[0165] A metal salt capable of forming a water-insoluble salt withphosphoric acid added may be added in a molar ratio of 0.1 to 10, morepreferably 0.5 to 5 to a 1-phosphorylated saccharide derivative used inthe reaction. There are no restrictions to an addition procedure of thesalt, and it may be added in one portion or portionwise during thereaction. This invention basically uses water as a solvent, but anorganic solvent such as an alcohol and dimethylsulfoxide used in acommon enzyme reaction may be, if necessary, added in an appropriateamount. In a reaction with a higher concentration, a base as a substrateor a nucleoside as a product may be not be completely dissolved in thereaction solution. This invention may be also applied to such a case.

[0166] A nucleoside compound produced as described above may be isolatedby a common procedure such as concentration, crystallization,dissolution, electrodialysis and adsorption and desorption using anion-exchange resin or charcoal.

EXAMPLES

[0167] This invention will be more specifically described with referenceto, but not limited to, Examples.

Example 1

[0168] Preparation of an Anomer Mixture of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribose-1-phosphate (18) andbis[3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribos-1-yl]phosphate (19)

[0169] To a mixture of 1.18 g of orthophosphoric acid in 51 mL ofacetonitrile were added 2.3 g of tri-n-butylamine and 5.07 g ofmolecular sieves 4A, and the mixture was cooled to 5° C. with stirring.After one hour, to the mixture was added 5.07 g of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-ribosyl chloride (purity: 85%),and the mixture was stirred for one hour to give a solution of a mixtureof the title compounds (18) and (19) [(18):(19)=3:5, α-form/β-form ofcompound (18)=5/2] in acetonitrile.

[0170] For preparing a sample for analysis, these compounds wereconverted into cyclohexylamine salts, which were then purified by silicagel column chromatography to provide two anomer isomers (19a) and (19b)of the title compound (19) from a fraction eluted with methanol-ethylacetate (1:10). (19a): Less polar fraction

[0171]¹H NMR (CDCl₃, 270 MHz) d: 8.0-7.8 (m, 8H), 7.4-7.2 (m, 8H), 6.06(m, 1.2H), 5.98 (m, 0.8H), 5.56 (m, 1.2H), 5.41 (m, 0.8H), 4.7-4.3 (m,6H), 2.6-2.4 (m, 1H), 2.75-2.6 (m, 2H), 2.5-2.3 (m, 2H), 2.2-1.9 (m,2H), 1.8-1.6 (m, 2H), 1.6-0.9 (m, 8H); MS (APCI) m/z 883 (M−H). (19b):More polar fraction

[0172]¹H NMR (CDCl₃, 270 MHz) d: 8.0-7.8 (m, 8H), 7.4-7.2 (m, 8H),6.1-5.9 (m, 2H), 5.55 (m, 0.67H), 5.39 (m, 1.33H), 4.7-4.3 (m, 6H),3.1-2.85 (m, 1H), 2.75-2.4 (m, 2H), 2.32 (m, 2H), 2.2-1.9 (m, 2H),1.8-1.6 (m, 2H), 1.6-0.9 (m, 8H); MS (APCI) m/z 883 (M−H).

Example 2

[0173] Preparation of an Anomer Mixture of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribose-1-phosphate andbis[3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribos-1-yl]phosphate

[0174] To a mixture of 1.11 g of orthophosphoric acid in 49 mL of2-butanone were added 2.11 g of tri-n-butylamine and 4.9 g of molecularsieves 4A, and the mixture was cooled to 5° C. with stirring. To themixture was added 4.9 g of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-ribosyl chloride (purity: 85%),and the mixture was stirred for 10 min to give a solution of a mixtureof the title compounds (18) and (19) [(18): (19)=1:4, α-form/β-form ofcompound (18)=7/10] in 2-butanone.

Example 3

[0175] Preparation of an Anomer Mixture of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribose-1-phosphate andbis[3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribos-1-yl]phosphate

[0176] To a mixture of 136.8 g of orthophosphoric acid in 2 L of2-butanone were added 90.6 g of tri-n-butylamine and 200 g of molecularsieves 4A, and the mixture was cooled to 5° C. with stirring. Afterstirring for one hour, to the mixture was added 200 g of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-a-D-ribosyl chloride (purity: 85%),and the mixture was stirred for 2 hours to give a solution of a mixtureof the title compounds (18) and (19) [(18):(19)=5:4, α-form/β-form ofcompound (18)=5/2] in 2-butanone.

Example 4

[0177] Preparation of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-ribose-1-phosphate (18a)

[0178] The acetonitrile solution prepared in Example 1 was cooled to 5°C. with stirring, and 2.29 g of orthophosphoric acid was added to themixture. After stirring for 3 hours, crystallization was initiated andthen the mixture became a thick suspension. After 5 hours, the ratio ofα-form/β-form of the title compound (18) in the reaction suspension was10/1. The crystals were collected as a mixture with molecular sieves.The solid was dissolved in 100 mL of methanol and the mixture was againfiltrated to remove molecular sieves. HPLC assay showed that 3.68 g ofthe title compound (18a) was contained in the methanol solution (Yield:74.6% after reduction from the purity of the starting material) withoutthe β-form on HPLC.

Example 5

[0179] Preparation of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-robose-1-phosphate (18a)

[0180] The 2-butanone solution prepared in Example 2 was cooled to 5° C.with stirring, and 2.2 g of orthophosphoric acid was added to thesolution. After stirring for 1 hour, precipitation of crystals initiatedand then a thick suspension was obtained. After 20 hours, the ratio ofα-form/β-form for compound (18a) in the reaction suspension was 8:1. Tothe suspension was added 6.33 g of tri-n-butylamine to dissolve theprecipitated crystals and molecular sieves were removed by filtration.To the filtrate was added 250 mL of toluene, and the solution was washedwith 55 mL of water. The organic layer was ice-cooled. To the mixturewas added 2.32 g of cyclohexylamine for crystallization with stirring.After 1 hour, the precipitated crystals were collected by filtration anddried in vacuo at room temperature to provide 3.19 g of adicyclohexylamine salt of compound (16a) as a colorless powder (Yield:64.7% after reduction from the purity of the starting material;α-form:β-form=97.5:2.5).

[0181]¹H NMR (DMSO-d₆, 270 MHz) d: 8.00 (d, J=8.6 Hz, 2H), 7.96 (d,J=8.6 Hz, 2H), 7.58 (d, J=8.6 Hz, 2H), 7.58 (d, J=8.6 Hz, 2H), 5.82 (dd,J=5.3, 5.3 Hz, 1H), 5. 36 (d, J=8.6 Hz, 1H), 4.6-4.3 (m, 3H), 4.7-3.5(br, 6H), 2.7-2.6 (m, 2H), 2.55-2.4 (m, 1H), 2.25 (d, J=4.2 Hz, 1H),1.85-1.75 (m, 4H), 1.7-1.6 (m, 4H), 1.55-1.45 (m, 2H), 1.25-0.9 (m,10H); MS (APCI) m/z 590 (M+C₆H₁₄N).

Example 6

[0182] Preparation of3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-robose-1-phosphate (18a)

[0183] The 2-butanone solution prepared in Example 3 was cooled to 5° C.with stirring. After stirring for 1 hour, precipitation of crystalsinitiated and then a thick suspension was obtained. After 23 hours, theratio of α-form/β-form for compound (18a) in the reaction suspension was7:1. To the suspension was added 259 g of tri-n-butylamine to dissolvethe precipitated crystals and molecular sieves were removed byfiltration. The filtrate was washed with 2.2 L of water and the aqueouslayer was extracted with 1 L of toluene. The combined organic layer wasice-cooled. To the mixture was added 87.5 g of cyclohexylamine forcrystallization with stirring. After 1 hour, the precipitated crystalswere collected by filtration and dried in vacuo at room temperature toprovide 213 g of a dicyclohexylamine salt of compound (16a) as acolorless powder (Yield: 78.1% after reduction from the purity of thestarting material; α-form:β-form=96.9:3.1).

Example 7

[0184] Preparation of 2-deoxy-α-D-ribose-1-phosphate (20)

[0185] To the methanol solution prepared in Example 4 was added 20 mL ofan aqueous solution of ammonium hydroxide, and the mixture was stirredat room temperature. After stirring for 28 hours, the precipitatedcrystals were collected by filtration and dried in vacuo at roomtemperature to provide 589 mg of an ammonium salt of compound (20) as acolorless powder (Yield: 21.1% without the β-form on HPLC).

Example 8

[0186] Preparation of 2-deoxy-α-D-ribose-1-phosphate (20)

[0187] Compound (18a) prepared in Example 6 was suspended in a mixtureof 2.3 L of methanol and 450 mL of an aqueous ammonium hydroxidesolution, and the mixture was stirred at room temperature. Afterstirring for 28 hours, the precipitated crystals were collected byfiltration and dried in vacuo at room temperature to provide 62.0 g ofan ammonium salt of compound (20) as a colorless powder (Yield: 81.0%without the β-form on HPLC).

[0188]¹H NMR (D₂O, 270 MHz) d: 5.56 (s, 1H), 4.03 (m, 2H), 3.52 (dd,J=3.3, 12.2 Hz, 1H), 3.41 (dd, J=5.3, 12.2 Hz, 1H), 2.17 (m, 1H), 1.87(d, J=13.9 Hz, 1H); MS (APCI) m/z: 213 (M−H).

Example 9

[0189] Preparation of2,3,5-O-tris(4-chlorobenzoyl)-α-D-ribose-1-phosphate (21)

[0190] To a mixture of 3.32 g of orthophosphoric acid in 67 mL of methylisobutyl ketone were added 2.11 g of tri-n-butylamine and 6.6 g ofmolecular sieves 4A, and the mixture was cooled to 5° C. with stirring.To the mixture was added 6.66 g of2,3,5-O-tris(4-chlorobenzoyl)-a-D-ribosyl chloride. After 1 hour,precipitation of crystals initiated and then a thick suspension wasprovided. After 10 hours, the ratio of α-form/β-form for compound (19)in the reaction suspension was 10:1. To the suspension was added 6.33 gof tri-n-butylamine to dissolve the precipitated crystals and molecularsieves were removed by filtration. The filtrate was washed with 55 mL ofwater. The organic layer was ice-cooled. To the mixture was added 2.4 gof cyclohexylamine for crystallization with stirring. After 1 hour, theprecipitated crystals were collected by filtration and dried in vacuo atroom temperature to provide 7.02 g of a dicyclohexylamine salt ofcompound (21) as a colorless powder (Yield: 73.0%; α-form:β-form =99:1).

[0191]¹H NMR (DMSO-d₆, 270 MHz) d: 8.2-7.8 (m, 6H), 7.6-7.4 (m, 6H),5.9-5.7 (m, 1H), 5.6-5.4 (m, 3H), 4.6-4.3 (m, 1H), 4.7-3.5 (br, 6H),2.7-2.6 (m, 2H), 1.9-1.7 (m, 4H), 1.7-1.6 (m, 4H), 1.55-1.4 (m, 2H),1.3-0.9 (m, 10H); MS (APCI) m/z 745 (M+C₆H₁₄N).

Example 10

[0192] Preparation of α-D-ribose-1-phosphate (22)

[0193] Compound (21) prepared in Example 9 was suspended in a mixture of105 mL of methanol and 21 mL of an aqueous ammonium hydroxide solution,and the mixture was stirred at room temperature. After stirring for 32hours, the precipitated crystals were collected by filtration and driedin vacuo at room temperature to provide 1.90 g of an ammonium salt ofcompound (22) as a colorless powder (Yield: 86.0% without the β-form onHPLC).

[0194]¹H NMR (D₂O, 270 MHz) d: 5.6 (m, 1H), 4.2 (m, 1H), 4.1-4.0 (m,2H), 3.75 (m, 1H), 3.7 (m, 1H); MS (APCI) m/z: 229 (M−H).

Example 11

[0195] Preparation of5-O-(4-chlorobenzoyl)-2,3-dideoxy-α-D-ribose-1-phosphate (23)

[0196] To a mixture of 3.5 g of orthophosphoric acid in 33 mL ofacetonitrile were added 2.2 g of tri-n-butylamine and 3.3 g of molecularsieves 4A, and the mixture was cooled to 5° C. with stirring. To themixture was added 3.28 g of5-O-(4-chlorobenzoyl)-2,3-dideoxy-a-D-ribosyl chloride. After 1 hour,precipitation of crystals initiated and then a-thick suspension wasprovided. After 20 hours, the ratio of α-form/β-form for compound (23)in the reaction suspension was 10:1. To the suspension was added 6.5 gof tri-n-butylamine to dissolve the precipitated crystals and molecularsieves were removed by filtration. The filtrate was diluted with 70 mLof toluene and then washed with 55 mL of water. The organic layer wasice-cooled. To the mixture was added 2.5 g of cyclohexylamine forcrystallization with stirring. After 1 hour, the precipitated crystalswere collected by filtration and dried in vacuo at room temperature toprovide 4.56 g of a dicyclohexylamine salt of compound (23) as acolorless powder (Yield: 71.5%; α-form:β-form=97:3).

[0197]¹H NMR (DMSO-d₆, 270 MHz) d: 8.2-7.8 (m, 2H), 7.6-7.4 (m, 2H),5.9-5.7 (m, 1H), 5.6-5.4 (m, 1H), 4.6-4.3 (m, 1H), 4.7-3.5 (br, 6H),2.7-2.6 (m, 2H), 1.9-1.7 (m, 8H), 1.7-1.6 (m, 4H), 1.55-1.4 (m, 2H),1.3-0.9 (m, 10H); MS (APCI) m/z 374 (M+C₆H₁₄N).

Example 12

[0198] Preparation of 2,3-dideoxy-α-D-ribose-1-phosphate (24)

[0199] Compound (23) prepared in Example 11 was suspended in a mixtureof 46 mL of methanol and 10 mL of an aqueous ammonium hydroxidesolution, and the mixture was stirred at room temperature. Afterstirring for 30 hours, the precipitated crystals were collected byfiltration and dried in vacuo at room temperature to provide 1.68 g ofan ammonium salt of compound (24) as a colorless powder (Yield: 85.0%without the β-form on HPLC).

[0200]¹H NMR (D₂O, 270 MHz) d: 5.2 (m, 1H), 4.1-3.9 (m, 1H), 3.6-3.3 (m,2H), 2.1-2.3 (m, 2H), 1.9-1.7 (m, 2H); MS (APCI) m/z: 197 (M−H).

Example 13

[0201] Preparation of 2,3,5-O-tris(4-chlorobenzoyl)-α-D-arabinofuranosyl-1-phosphate (25)

[0202] To a mixture of 3.3 g of orthophosphoric acid in 67 mL of methylisobutyl ketone were added 2.1 g of tri-n-butylamine and 6.6 g ofmolecular sieves 4A, and the mixture was cooled to 5° C. with stirring.To the mixture was added 6.6 g of2,3,5-O-tris(4-chlorobenzoyl)-α-D-arabinofuranosyl chloride. After 1hour, precipitation of crystals initiated and then a thick suspensionwas provided. After 8 hours, the ratio of α-form/β-form for compound(25) in the reaction suspension was 10:1. To the suspension was added6.3 g of tri-n-butylamine to dissolve the precipitated crystals andmolecular sieves were removed by filtration. The filtrate was washedwith 55 mL of water. The organic layer was ice-cooled. To the mixturewas added 2.4 g of cyclohexylamine for crystallization with stirring.After 1 hour, the precipitated crystals were collected by filtration anddried in vacuo at room temperature to provide 6.72 g of adicyclohexylamine salt of compound (25) as a colorless powder (Yield:70.5%; α-form:β-form=99:1).

[0203] MS (APCI) m/z 745 (M+C₆H₁₄N).

Example 14

[0204] Preparation of α-D-arabinofuranosyl-1-phosphate (26)

[0205] Compound (25) prepared in Example 13 was suspended in a mixtureof 94 mL of methanol and 18 mL of an aqueous ammonium hydroxidesolution, and the mixture was stirred at room temperature. Afterstirring for 48 hours, the precipitated crystals were collected byfiltration and dried in vacuo at room temperature to provide 1.72 g ofan ammonium salt of compound (26) as a colorless powder (Yield: 82.0%without the β-form on HPLC).

[0206]¹H NMR (D₂O, 270 MHz) d: 5.3 (m, 1H), 3.95-3.3 (m, 5H); MS (APCI)m/z: 229 (M−H).

Example 15

[0207] Preparation of (2R)-2-benzyloxymethyl-1,3-dioxorane-4-phosphate(27)

[0208] To a solution of 1.06 g of(2R)-2-benzyloxymethyl-4-(R,S)-acetoxy-1,3-dioxorane in 12 mL of etherunder ice-cooling was added 4 mL of a 4N solution of hydrochloric acidin dioxane. After stirring 3.5 hours, the mixture was warmed to roomtemperature. After removing the solvent by concentration, the residuewas further subject to azeotropy with toluene to give 500 mg of(2R)-2-benzyloxymethyl-1,3-dioxoranyl chloride as a colorless andtransparent oil. To 1.1 mL of acetonitrile were sequentially added 0.27g of orthophosphoric acid, 0.66 mL of tri-n-butylamine and 0.23 g ofmolecular sieves 4A, and the mixture was stirred for 1.5 hours. To thesuspension under ice-cooling was added 0.27 g of the previous oil, andthe mixture was stirred under ice-cooling for 5.5 hours. To the mixturewas added 0.6 mL of tri-n-butylamine. After stirring for 30 min, themixture was diluted with toluene and extracted with water. The aqueouslayer was extracted with n-butanol and then concentrated. Theconcentrate was dissolved in toluene, and to the solution was addedcyclohexylamine to give a cyclohexylamine salt of compound (27) as awhite solid.

[0209]¹H-NMR (D₂O) δ: 0.98-1.10 (2H, m), 1.14-1.23 (6H, m), 1.47-1.51(2H, m), 1.61-1.64 (4H, m), 1.78-1.83 (4H, m), 2.94-3.00 (2H, m),3.46-3.60 (2H, m), 3.72-3.79 (1H, m), 3.92-4.00 (1H, m), 4.41-4.51 (2H,m), 5.01-5.03 and 5.22-5.24 (total 1H, m), 5.64-5.72 (total 1H, m),7.24-7.30 (5H, m); MS (APCI) m/z: 390 (M+C₆H₁₄N)+.

Example 16

[0210] Preparation of (2R)-2-hydroxymethyl-1,3-dioxorane-4-phosphate(28)

[0211] In 10 mL of methanol was dissolved 0.2 g of compound (27)prepared in Example 15. The solution was subject to hydrogenation underan ambient pressure using 0.11 g of 10% Pd/C as a catalyst. Afterremoving the catalyst by filtration, the filtrate was concentrated togive a cyclohexylamine of compound (28).

[0212]¹H-NMR (D₂O) δ: 0.99-1.06 (2H, m), 1.10-1.24 (6H, m), 1.47-1.50(2H, m), 1.62-1.66 (4H, m), 1.80-1.85 (4H, m), 1.96-3.02 (2H, m),3.51-3.57 (2H, m), 3.72-3.79 (1H, m), 3.93-4.00 (1H, m), 4.99-5.01 and5.13-5.15 (total 1H, m), 5.64-5.67 and 5.70-5.73 (total 1H, m); MS(APCI) m/z: 199 (M−H)⁻.

Example 17

[0213] Preparation of2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-α-D-erythropentofuranose-1-phosphate(29)

[0214] Seventy mg of molecular sieves 4A was added to a stirred mixtureof 62 mg of orthophosphoric acid, 52 μL of tri-n-butylamine and 0.7 mLof acetonitrile at room temperature, and the mixture was stirred in anice-bath. To the mixture was added 70 mg of2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-D-erythropentofuranosylchloride, and the mixture was reacted at the same temperature for 1 day.Then, to the mixture were added 156 μL of tri-n-butylamine and thendeionized water. The mixture was extracted with toluene three times. Tothe organic layer was added 48 μL of cyclohexylamine and the mixture wasstirred for 30 min. The mixture was concentrated in vacuo, and acetonewas added to form a precipitate, which was collected by filtration. Theresidue was washed with chloroform and dried in vacuo at roomtemperature to give a dicyclohexylamine of compound (29) as a whitesolid.

[0215]¹H-NMR (CD₃OD) δ: 1.1-1.4 (10H, m), 1.65 (2H, m), 1.89 (4H, m),1.96 (4H, m), 2.3-2.5 (2H, m), 2.91 (2H, m), 4.5 (2H, m), 4.6-4.8 (1H,m), 5.1-5.3 (1H, m), 5.97 (1H, m), 7.41 (1H, m), 7.47 (2H, m), 7.68 (2H,m), 7.75 (2H, m), 8.08 (2H, m); MS (APCI) m/z: 496 (M+C₆H₁₄N)⁺.

Example 18

[0216] Preparation of2,3-dideoxy-3-fluoro-α-D-erythropentofuranose-1-phophate (30)

[0217] To a solution of 21 mg of compound (29) prepared in Example 17 in1 mL of methanol was added 20 μL of cyclohexylamine and the mixture wasreacted for 2 weeks. The mixture was concentrated in vacuo and diethylether was added. The mixture was filtered, and the solid was dried invacuo to give 12 mg of a dicyclohexylamine salt of the title compound asa white solid.

[0218]¹H-NMR (CD₃OD) δ: 1.1-1.4 (10H, m), 1.66 (2H, m), 1.79 (4H, m),1.94 (4H, m), 2.3-2.4 (2H, m), 2.88 (2H, m), 3.59 (2H, m), 4.3-4.4 (1H,m), 5.11 (0.5H. m; the other 0.5H was undistinguishable because it wasbehind the peak of water), 5.89 (1H, m); MS (APCI) m/z: 215 (M−H)⁻.

Example 19

[0219] Preparation of2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-D-erythropentofuranose-1-phosphate(31)

[0220] At room temperature 0.86 g of molecular sieves 4A was added to astirred mixture of 759 mg of orthophosphoric acid, 646 μL oftri-n-butylamine and 8.6 mL of acetonitrile at room temperature, and themixture was stirred in an ice-bath. To the mixture was added 864 mg of2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-D-erythropentofuranosylchloride, and the mixture was reacted at the same temperature for 1 day.Then, to the mixture were added 1.94 mL of tri-n-butylamine and thendeionized water. The mixture was extracted with toluene three times andwashed with purified water five times. The organic layer was separated.To the organic layer was added 590 μL of cyclohexylamine and the mixturewas stirred for 30 min. The mixture was concentrated in vacuo. Afteraddition of acetone, the mixture was stirred and filtrated. The residuewas further washed with isopropyl ether and dried in vacuo at roomtemperature to give compound (31) as a white solid. α-form:β-form=66:34.

[0221]¹H-NMR (CD₃OD) δ: 1.1-1.4 ppm (10H, m), 1.66 (2H, m), 1.78 (4H,m), 1.98 (4H, m), 2.3-2.6 (2H, m), 2.89 (2H, m), 4.44 & 4.46 (a & 1,2H),4.6-4.8 (1H, m), 5.1-5.3 & 5.3-5.4 (a & 1,1H, m), 5.97 & 6.00 (a & 1,1H,m), 7.40 (1H, m), 7.47 (2H, m), 7.68 (2H, m), 7.75 (2H, m), 8.07 (1H,m), 8.13 (1H, m).

Example 20

[0222] Preparation of 2,3-dideoxy-3-fluoro-D-erythropentofuranose-1-phophate (32)

[0223] To a solution of 0.29 g of compound (31) prepared in Example 19in 15 mL of methanol was added 279 μL of cyclohexylamine and the mixturewas reacted for 1 week. The mixture was concentrated in vacuo anddiethyl ether was added. After stirring, the mixture was filtered, andthe solid was dried in vacuo to give 185 mg of a dicyclohexylamine saltof compound (32) as a white solid. α-form:β-form=66:34.

[0224]¹H-NMR (CD₃OD) δ: 1.1-1.4 ppm (10H, m), 1.67 (2H, m), 1.79 (4H,m), 2.2-2.4 (2H, m), 2.94 (2H, m), 3.59 & 3.62 (a & 1,2H, m), 3.3-3.4(2H, m), 5.10 & 5.1-5.24 (α & β, 0.5H & 1H, m, 0.5H of the a-form wasundistinguishable because the signal was behind the peak of water), 5.88& 5.93 (α & β, 1H, m).

Example 21

[0225] Preparation of 3,5-O-dibenzoyl-2-O-methylribose-1-phosphate (33)

[0226] To 2.84 g of 1,3,5-O-tribenzoyl-2-O-methyl-α-D-ribose was added14.5 mL of a 4N solution of hydrochloric acid in dioxane, and themixture was stirred under ice-cooling. After stirring 2.5 hours, 10 mLof a 4N solution of hydrochloric acid in dioxane was further added, andthe mixture stirred for 1 hour. After evaporating the solvent, theresidue was further subject to azeotropy with 10 mL of dioxane twice togive 3,5-O-dibenzoyl-2-O-methylribosyl-1-chloride. Separately, 2.98 g of98% phosphoric acid was dissolved in 15 mL of 4-methyl-2-pentanone andafter adding 2.8 g of molecular sieves 4A, the mixture was stirred for30 min. To the mixture were added 1.42 mL of tri-n-butylamine and then asolution of the previous 3,5-O-dibenzoyl-2-O-methylribosyl-1-chloride in10 mL of 4-methyl-2-pentanone. After reacting the mixture at roomtemperature for 20 hours, it was neutralized with 7.1 mL oftri-n-butylamine. After removing the molecular sieves by filtration, thefiltrate was washed with 20 mL of water three times. The organic layerwas evaporated and purified by silica gel column chromatography to give950 mg of compound (33).

[0227] MS (APCI) m/z: 451 (M−H)⁻; IR (KBr) cm⁻¹: 3448, 2963, 1721, 1453,1278, 1111, 976, 711, 558.

Example 22

[0228] Preparation of 2-O-methylribose-1-β-phosphate (34)

[0229] To 850 mg of compound (33) prepared in Example 21 was 20 mL of14% ammonia-methanol, and the mixture was reacted at room temperaturefor 20 hours. After evaporation of the solvent, diisopropyl ether wasadded to form a sludge and the crystalline powder was collected byfiltration. The powder was dissolved in methanol. To the solution wasadded cyclohexylamine, and the mixture was stirred. After evaporatingmethanol, diisopropyl ether was added to the residue to form a sludge.The crystalline powder was collected by filtration and washed withdiisopropyl ether. The desired product was extracted with water and theaqueous layer was washed with 4-methyl-2-pentanone twice. The aqueouslayer was concentrated and to the layer was added diisopropyl ether toform a sludge. After filtration, the crystals were washed withdiisopropyl ether to give 120 mg of a dicyclohexylamine salt of compound(34).

[0230]¹H-NMR (D₂O) δ: 3.37 (s, 3H), 3.49 (dd, 1H, J=4.9 Hz, 12.7 Hz),3.62 (d, 1H, J=4.9 Hz), 3.69 (dd, 1H, J=2.7 Hz, 12.7 Hz), 3.74-3.78 (m,1H), 4.28 (dd, 1H, J=4.6 Hz, 7.8 Hz), 5.39 (d, 1H, J=5.9 Hz); MS (APCI)m/z: 243 (M−H)—.

Example 23

[0231] Preparation of 3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-ribose-1-phosphate (18a)

[0232] To a mixture of 6.92 g of orthophosphoric acid in 80 mL ofacetonitrile were added 5.51 mL of tri-n-butylamine and 10 g ofmolecular sieves 4A. The mixture was stirred at room temperature for 5hours and allowed to stand overnight. After cooling to −7° C., to themixture was added 10 g of 3,5-O-bis(4-chlorobenzoyl)-2-deoxy-α-D-ribosylchloride (purity: 85%). The mixture was stirred for 9 hours and allowedto stand at −15° C. overnight. After adding 16.5 mL of tri-n-butylamine,the molecular sieves were removed by filtration. The filtrate wasconcentrated and the residue was dissolved in 4-methyl-2-pentanone andwashed with water. The organic layer was ice-cooled and 5.66 mL ofcyclohexylamine was added with stirring for crystallization. After 1.5hours, the precipitated crystals were filtered and dried in vacuo atroom temperature to give 13.5 g of a dicyclohexylamine salt of compound(18a). α-form:β-form=98.8:1.2).

Example 24

[0233] Preparation of 2-deoxy-a-D-ribose-1-phosphate (20)

[0234] To a solution of 7.05 g of the compound obtained in Example 23 inmethanol was added 2.92 mL of cyclohexylamine, and the mixture wasstirred at room temperature. After stirring 72 hours, the mixture wasconcentrated and to the residue was added ethanol to provide asuspension which was then stirred. After collecting the precipitatedcrystals, they were dried I vacuo at room temperature to give 3.87 g ofa dicylcohexylamine salt of compound (20) (without the β-form on NMR).

[0235]¹H NMR (D₂O) d: 5.57 (dd, J=5.1, 6.1 Hz, 1H), 4.03 (m, 2H), 3.54(ddd, J=1.2, 2.2, 12.2 Hz, 1H), 3.42 (ddd, J=1.2, 5.1, 12.2 Hz, 1H),3.18-2.94 (m, 2H), 2.17 (m, 1H), 1.90 (d, J=1.2, 12.8 Hz, 1H), 1.8-1.45(m, 10H), 1.25-0.9 (m, 12H).

[0236] Anal. Calcd. for C₅H₉O₇P.C₁₂H₂₈N₂, C: 49.50%; H: 9.04%; N: 6.79%;P: 7.51%,

[0237] Found C: 49.26%; H: 8.81%; N: 6.64%; P: 7.29%.

Example 25

[0238] Preparation of 2′-deoxyadenosine (1)

[0239] Fifty mL of an LB medium was inoculated with Escherichia coliK-12/XL-10 strain (Stratagene Inc.) and it was cultured at 37° C.overnight. After collection, the bacteria was lysed with a lysissolution containing 1 mg/mL of lysozyme. The lysis solution was treatedwith phenol and DNA was precipitated as usual by ethanol precipitation.The DNA precipitate was collected with a glass rod and washed to preparean E. coli chromosome DNA.

[0240] Oligonucletides of SEQ ID Nos. 1 and 2 designed based on thesequence a known deoD gene in Escherichia coli (GenBank accession No.AE000508 with a coding region of base numbers 11531 to 12250) were usedas primers for PCR. These primers have restriction enzyme recognitionsequences for EcoRI and Hind III near 5′- and 3′-ends, respectively.

[0241] SEQ ID No. 1: GTGAATTCAC AAAAAGGATA AAACAATGGC

[0242] SEQ ID No. 2: TCGAAGCTTG CGAAACACAA TTACTCTTT

[0243] Using 0.1 mL of a PCR reaction solution containing 6 ng/μL of theabove E. coli chromosome DNA completely digested by restriction enzymeHind III and the primers (each at 3 μM), PCR was conducted by 30 cyclesunder the conditions of denaturation: 96° C., 1 min; annealing: 55° C.,1 min; elongation: 74° C., 1 min per a cycle.

[0244] The above reaction product and a plasmid pUC18 (Takara Shuzo Co.Ltd.) were digested by EcoRI and Hind III and ligated usingLigation-High (Toyobo Co. Ltd.). The recombinant plasmid obtained wasused to transform Escherichia coli DH5α. The transformed strain wascultured in an LB agar medium containing 50 μg/mL of ampicillin andX-Gal (5-bromo-4-chloro-3-indolyl-β-galactoside) to provide anAm-resistant transformant as a white colony. A plasmid was extractedfrom the transformant thus obtained and the plasmid in which a desiredDNA fragment had been inserted was designated as pUC—PNP73. Thetransformant thus obtained was designated as Escherichia coli MT-10905.

[0245]Escherichia coli MT-10905 was cultured by shaking at 37° C.overnight in 100 mL of an LB medium containing 50 μg/mL of Am. Theculture medium was centrifuged at 13,000 rpm for 10 min to collect thebacteria. The bacteria were suspended in 10 mL of 10 mMTris-hydrochloride buffer (pH 8.0) and ultrasonicated to give ahomogenate which was then used as an enzyme source.

[0246] Reaction solutions were prepared by adding calcium chloride (WacoPure Chemicals, Extra pure grade) at different concentrations to amixture of 2.5 mM 2-deoxy-α-D-ribose-1-phosphate diammonium saltprepared in Example 8, 2.5 mM adenine (Wako Pure Chemicals, Extra puregrade), 0.1 mL of the ultrasonic enzyme homogenate from apurinenucleoside-phosphorylase producing strain and 10 mMTris-hydrochloride buffer (pH 7.4). One mL of a reaction solution wasreacted at 30° C. for 24 hours. At the end of the reaction, a whiteprecipitate had been formed.

[0247] HPLC analysis described below for a post-reaction solution showeda peak completely identical to the peak of 2′-deoxyadenosine (Wako PureChemicals, Extra pure grade) in all the post-reaction solutions.

[0248] HPLC Analysis Conditions

[0249] Column: YMC-Pack ODS—A312, 150×6.0 mm I.D.

[0250] Column temperature: 40° C.

[0251] Pump flow rate: 0.75 mL/min

[0252] Detection: UV 260 nm

[0253] Eluent: 10 mM phosphoric acid:acetonitrile=95:5 (V/V)

[0254] Table 1 shows the calculation results of a reaction inversionrate after determining a concentration of 2′-deoxyadenosine in apost-reaction solution. TABLE 1 Amount of calcium Reaction inversionchloride (mM) rate (%) 0.0 80.4 2.5 90.8 10.0 96.0

Example 26

[0255] Preparation of 2′-deoxyadenosine (2)

[0256] A reaction was conducted as described in Example 25 except thataluminum chloride was added in place of calcium chloride. At the end ofthe reaction, a white precipitate had been formed. HPLC analysis for thepost-reaction solutions as described in Example 25 showed a peakcompletely identical to the peak of 2′-deoxyadenosine (Wako PureChemicals, Extra pure grade) in all the post-reaction solutions. Table 2shows the calculation results of a reaction inversion rate afterdetermining a concentration of 2′-deoxyadenosine in a post-reactionsolution. TABLE 2 Amount of calcium Reaction inversion chloride (mM)rate (%) 0.0 80.4 (same as in Example 15) 2.5 90.2 10.0 93.3

Example 27

[0257] Preparation of 2′-deoxyadenosine (3)

[0258] A reaction was conducted as described in Example 25 except that10 mM of barium chloride was added in place of calcium chloride. At theend of the reaction, a white precipitate had been formed. HPLC analysisfor the post-reaction solutions as described in Example 25 showed a peakcompletely identical to the peak of 2′-deoxyadenosine (Wako PureChemicals, Extra pure grade) in the post-reaction solutions. A reactioninversion rate after determining a concentration of 2′-deoxyadenosine ina post-reaction solution was calculated to be 92.4%.

Example 28

[0259] Preparation of Thymidine

[0260] One mL of a reaction solution consisting of 2.5 mM2-deoxy-α-D-ribose-1-phosphate diammonium salt prepared in Example 8,2.5 mM thymine (Wako Pure Chemicals, Extra pure grade), 12 units/mLthymidine phosphorylase (SIGMA), 0 mM or 10 mM calcium nitrate (WakoPure Chemicals, Extra pure grade) and 10 mM Tris-hydrochloride buffer(pH 7.4) was reacted at 30° C. for 24 hours. At the end of the reaction,a white precipitate had been formed. HPLC analysis for the post-reactionsolutions as described in Example 25 showed a peak completely identicalto the peak of thymidine (Wako Pure Chemicals, Extra pure grade) in thepost-reaction solution. Table 3 shows the calculation results of areaction inversion rate after determining a concentration of thymidinein the post-reaction solution. TABLE 3 Amount of calcium Amount ofthymidine chloride (mM) formed (mM) 0 75.2 10.0 91.2

Example 29

[0261] Preparation of 2′-deoxyadenosine (4)

[0262] One mL of a reaction solution consisting of 100 mM2-deoxy-α-D-ribose-1-phosphate diammonium salt prepared in Example 8,100 mM adenine (Wako Pure Chemicals, Extra pure grade), 0.1 mL of theultrasonic enzyme homogenate from a purinenucleoside-phosphorylaseproducing strain prepared in Example 25, 0 to 150 mM calcium chloride(Waco Pure Chemicals, Extra pure grade) and 100 mM Tris-hydrochloridebuffer (pH 8.0) was reacted at 50° C. for 24 hours. At the end of thereaction, a white precipitate had been formed. HPLC analysis for thepost-reaction solutions as described in Example 25 showed a peakcompletely identical to the peak of 2-deoxyadenosine (Wako PureChemicals, Extra pure grade) in the post-reaction solutions. Table 4shows the calculation results of determining a concentration of2′-deoxyadenosine in a post-reaction solution. TABLE 4 Amount of calciumAmount of 2′- chloride deoxyadenosine formed (mM) (mM) 0 85.0 20 90.0 6096.5 100 97.8 150 97.5

Example 30

[0263] Preparation of 2′-deoxyguanosine

[0264] One mL of a reaction solution consisting of 100 mM2-deoxy-a-D-ribose-1-phosphate diammonium salt prepared in Example 8,100 mM guanine (Wako Pure Chemicals, Extra pure grade), 0.1 mL of theultrasonic enzyme homogenate from a purinenucleoside-phosphorylaseproducing strain prepared in Example 25, 0 mM or 150 mM calcium chloride(Waco Pure Chemicals, Extra pure grade) and 100 mM Tris-hydrochloridebuffer (pH 8.0) was reacted at 50° C. for 24 hours. At the end of thereaction, a white precipitate had been formed. HPLC analysis for thepost-reaction solution as described in Example 25 showed a peakcompletely identical to the peak of 2-deoxyguanosine (Wako PureChemicals, Extra pure grade) in the post-reaction solution. Table 5shows the calculation results of determining a concentration of2′-deoxyguanosine in the post-reaction solution. TABLE 5 Amount ofcalcium Amount of 2′-deoxy- chloride (mM) guanosine formed (mM) 0 50.0150 97.5

Example 31

[0265] Preparation of Adenosine

[0266] One mL of a reaction solution consisting of 100 mMa-D-ribose-1-phosphate diammonium salt prepared in Example 10, 100 mMadenine (Wako Pure Chemicals, Extra pure grade), 0.1 mL of theultrasonic enzyme homogenate from a purinenucleoside-phosphorylaseproducing strain prepared in Example 25, 0 mM or 150 mM calcium chloride(Waco Pure Chemicals, Extra pure grade) and 100 mM Tris-hydrochloridebuffer (pH 8.0) was reacted at 50° C. for 24 hours. At the end of thereaction, a white precipitate had been formed. HPLC analysis for thepost-reaction solution as described in Example 25 showed a peakcompletely identical to the peak of adenosine (Wako Pure Chemicals,Extra pure grade) in the post-reaction solution. Table 6 shows thecalculation results of determining a concentration of adenosine in thepost-reaction solution. TABLE 6 Amount of calcium Amount of adenosinechloride (mM) formed (mM) 0 86.1 150 98.4

Example 32

[0267] Preparation of 2′,3′-dideoxyadenosine

[0268] One mL of a reaction solution consisting of 100 mM2,3-dideoxy-α-D-ribose-1-phosphate diammonium salt prepared in Example12, 100 mM adenine (Wako Pure Chemicals, Extra pure grade), 0.1 mL ofthe ultrasonic enzyme homogenate from a purinenucleoside-phosphorylaseproducing strain prepared in Example 25, 0 mM or 150 mM calcium chloride(Waco Pure Chemicals, Extra pure grade) and 100 mM Tris-hydrochloridebuffer (pH 8.0) was reacted at 50° C. for 24 hours. At the end of thereaction, a white precipitate had been formed. HPLC analysis for thepost-reaction solution as described in Example 25 showed a peakcompletely identical to the peak of 2′,3′-dideoxyadenosine (Sigma, Extrapure grade) in the post-reaction solution. Table 7 shows the calculationresults of determining a concentration of 2′,3′-dideoxyadenosine in thepost-reaction solution. TABLE 7 Amount of calcium Amount of2,3′-dideoxy- chloride (mM) adenosine formed (mM) 0 82.4 150 96.4

Example 33

[0269] Preparation of adenine-9-β-D-arabinoside

[0270] One mL of a reaction solution consisting of 100 mMα-D-arabinofuranosyl-1-phosphate diammonium salt prepared in Example 14,100 mM adenine (Wako Pure Chemicals, Extra pure grade), 0.1 mL of theultrasonic enzyme homogenate from a purinenucleoside-phosphorylaseproducing strain prepared in Example 25, 0 mM or 150 mM calcium chloride(Waco Pure Chemicals, Extra pure grade) and 100 mM Tris-hydrochloridebuffer (pH 8.0) was reacted at 50° C. for 24 hours. At the end of thereaction, a white precipitate had been formed. HPLC analysis for thepost-reaction solution as described in Example 25 showed a peakcompletely identical to the peak of adenine-arabinoside (Sigma, Extrapure grade) in the post-reaction solution. Table 8 shows the calculationresults of determining a concentration of adenine-9-β-D-arabinoside inthe post-reaction solution. TABLE 8 Amount of calcium Amount ofadenine-9-β-D- chloride (mM) arabinoside formed (mM) 0 79.4 150 93.4

Example 34

[0271] Preparation of 2-amino-6-chloropurine-2′-deoxy-β-D-riboside

[0272] One mL of a reaction solution consisting of 10 mM2-deoxy-α-D-ribose-1-phosphate diammonium salt prepared in Example 8, 10mM 2-amino-6-chloropurine (Tokyo Kasei), 100 mM Tris-hydrochloridebuffer (pH 7.5) and 50 μL of the ultrasonic enzyme homogenate from apurinenucleoside-phosphorylase producing strain prepared in Example 25was reacted at 50° C. for 4 hours. At the end of the reaction, a whiteprecipitate had been formed. HPLC analysis for the post-reactionsolution under the conditions below showed a peak of2-amino-6-chloropurine-2′-deoxy-α-D-riboside. A reaction inversion ratewas calculated to be 20.9% after determining the concentration of2-amino-6-chloropurine-2′-deoxy-β-D-riboside in the post-reactionsolution.

[0273] HPLC Analysis Conditions

[0274] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0275] Column temperature: 40° C.

[0276] Pump flow rate: 1.0 mL/min

[0277] Detection: UV 254 nm

[0278] Eluent: 25 mM potassium dihydrogen phosphate:methanol=875:125(V/V)

Example 35

[0279] Preparation of 2,6-diaminopurine-2′-deoxy-β-D-riboside

[0280] A reaction was conducted as described in Example 34 except that2,6-diaminopurine (Tokyo Kasei) was added in place of2-amino-6-chloropurine. HPLC analysis for the post-reaction solution asdescribed in Example 34 showed a peak of2,6-diaminopurine-2′-deoxy-β-D-riboside. A reaction inversion rate wascalculated to be 75.5% after determining the concentration of2,6-diaminopurine-2′-deoxy-β-D-riboside in the post-reaction solution.

Example 36

[0281] Preparation of 6-mercaptopurine-2′-deoxy-β-D-riboside

[0282] A reaction was conducted as described in Example 34 except that6-mercaptopurine (KOUJIN) was added in place of 2-amino-6-chloropurine.HPLC analysis for the post-reaction solution as described in Example 34showed a peak of 6-mercaptopurine-2′-deoxy-β-D-riboside. A reactioninversion rate was calculated to be 57.2% after determining theconcentration of 6-mercaptopurine-2′-deoxy-β-D-riboside in thepost-reaction solution.

Example 37

[0283] Preparation of 2-amino-6-iodopurine-2¹-deoxy-β-D-riboside

[0284] A reaction was conducted as described in Example 34 except that2-amino-6-iodopurine was added in place of 2-amino-6-chloropurine. HPLCanalysis for the post-reaction solution as described in Example 34showed a peak of 2-amino-6-iodopurine-2′-deoxy-p-D-riboside. A reactioninversion rate was calculated to be 69.2% after determining theconcentration of 2-amino-6-iodopurine-2′-deoxy-β-D-riboside in thepost-reaction solution.

Example 38

[0285] Preparation of2-acetylamino-6-hydroxypurine-2′-deoxy-β-D-riboside

[0286] A reaction was conducted as described in Example 34 except that2-acetylamino-6-hydroxypurine (Tokyo Kasei) was added in place of2-amino-6-chloropurine. HPLC analysis for the post-reaction solutionunder the conditions described below showed a peak of2-acetylamino-6-hydroxypurine-2′-deoxy-β-D-riboside. A reactioninversion rate was calculated to be 48.7% after determining theconcentration of 2-acetylamino-6-hydroxypurine-2′-deoxy-β-D-riboside inthe post-reaction solution.

[0287] HPLC Analysis Conditions

[0288] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0289] Column temperature: 40° C.

[0290] Pump flow rate: 1.0 mL/min

[0291] Detection: UV 254 nm

[0292] Eluent: 25 mM potassium dihydrogen phosphate:methanol=75:25 (V/V)

Example 39

[0293] Preparation of2-amino-6-cyclopropylaminopurine-21-deoxy-β-D-riboside

[0294] A reaction was conducted as described in Example 34 except that2-amino-6-cyclopropylaminopurine was added in place of2-amino-6-chloropurine. HPLC analysis for the post-reaction solution asdescribed in Example 38 showed a peak of2-amino-6-cyclopropylaminopurine-2′-deoxy-β-D-riboside. A reactioninversion rate was calculated to be 87.6% after determining theconcentration of 2-amino-6-cyclopropylaminopurine-21-deoxy-β-D-ribosidein the post-reaction solution.

Example 40

[0295] Preparation of 2′,3′-dideoxy-3′-fluoro-D-guanosine

[0296] One mL of a reaction solution consisting of 7.0 mM2,3-dideoxy-3-fluoro-D-erythropentofuranose-1-phosphate prepared inExample 18, 10 mM guanine (Tokyo Kasei), 100 mM Tris-hydrochloridebuffer (pH 7.5) and 0.1 mL of the ultrasonic enzyme homogenate from apurinenucleoside-phosphorylase producing strain prepared in Example 25was reacted at 50° C. for 114 hours. HPLC analysis for the post-reactionsolution as described in Example 34 showed a peak of2′,3′-dideoxy-3′-fluoro-D-guanosine. A reaction inversion rate wascalculated to be 47.7% after determining the concentration of2′,3′-dideoxy-3¹-fluoro-D-guanosine in the post-reaction solution.

Example 41

[0297] Preparation of 2′,3′-dideoxy-3′-fluoro-D-guanosine

[0298] One mL of a reaction solution consisting of 7.0 mM2,3-dideoxy-3-fluoro-D-erythropentofuranose-1-phosphate prepared inExample 18, 10 mM guanine (Tokyo Kasei), 100 mM Tris-hydrochloridebuffer (pH 7.5) and 0.1 mL of the ultrasonic enzyme homogenate from apurinenucleoside-phosphorylase producing strain prepared in Example 25was reacted at 50° C. for 47 hours. To the solution was added calciumchloride to a final concentration of 20 mM and the mixture was reactedat 50° C. for additional 67 hours. HPLC analysis for the post-reactionsolution as described in Example 34 showed a peak of2′,3′-dideoxy-3′-fluoro-D-guanosine. A reaction inversion rate wascalculated to be 84.4% after determining the concentration of2′,3′-dideoxy-3′-fluoro-D-guanosine in the post-reaction solution.

Example 42

[0299] Preparation of 6-chloro-9-(β-D-ribofuranos-1-yl)purine

[0300] One mL of a reaction solution consisting of 10 mM 6-chloropurine(Aldrich), 50 mM D-ribose-1-phosphate (22) prepared in Example 10, 0.1mL of the ultrasonic enzyme homogenate from apurinenucleoside-phosphorylase producing strain prepared in Example 25and 100 mM Tris-hydrochloride buffer (pH 7.5) was reacted at 50° C. for20 hours. After completion of the reaction, HPLC analysis for thereaction solution under the conditions described below showed a peak ofthe title compound. A reaction inversion rate was calculated to be 62.4%after determining the concentration of6-chloro-9-(β-D-ribofuranos-1-yl)purine in the post-reaction solution.

[0301] HPLC Analysis Conditions

[0302] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0303] Column temperature: 40° C.

[0304] Pump flow rate: 1.0 mL/min

[0305] Detection: UV 254 nm

[0306] Eluent: 25 mM potassium dihydrogen phosphate:methanol=75:25 (V/V)

Example 43

[0307] Preparation of1-(2-deoxy-β-D-ribofuranos-1-yl)-1H-imidazo[4,5-b]pyridine and3-(2-deoxy-β-D-ribofuranos-1-yl)-1H-imidazo[4,5-b]pyridine

[0308] One mL of a reaction solution consisting of 10 mM2-deoxy-α-D-ribose-1-phosphate ammonium salt prepared in Example 8, 10mM 4-azabenzimidazole (Aldrich), 100 mM Tris-hydrochloride buffer (pH7.5) and 50 μL of the ultrasonic enzyme homogenate from apurinenucleoside-phosphorylase producing strain prepared in Example 25was reacted at 50° C. for 17 hours. HPLC analysis for the post-reactionsolution under the conditions described below showed two peaks of thetitle compounds. Reaction inversion rates were calculated to be 3% and7.2% after determining the concentrations of the products in thepost-reaction solution.

[0309] HPLC Analysis Conditions

[0310] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0311] Column temperature: 40° C.

[0312] Pump flow rate: 1.0 mL/min

[0313] Detection: UV 254 nm

[0314] Eluent: 25 mM potassium dihydrogen phosphate methanol=50:50 (V/V)

[0315] LC-MS analysis data: MS(APCI) m/z: 236 (MH)⁺

Example 44

[0316] Preparation of 8-aza-2′-deoxyadenosine

[0317] A reaction was conducted as described in Example 43 except that8-azaadenine (Aldrich) was used in place of 4-azabenzimidazole. HPLCanalysis for the post-reaction solution under the conditions describedbelow showed a peak of 8-aza-2′-deoxyadenosine. A reaction inversionrate was calculated to be 4.8% after determining the concentration of8-aza-2′-deoxyadenosine in the post-reaction solution.

[0318] HPLC Analysis Conditions

[0319] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0320] Column temperature: 40° C.

[0321] Pump flow rate: 1.0 mL/min

[0322] Detection: UV 254 nm

[0323] Eluent: 25 mM potassium dihydrogen phosphate methanol=875:125(V/V)

[0324] LC-MS analysis data: MS(APCI) m/z: 253 (MH)⁺

Example 45

[0325] Preparation of 8-aza-2′-deoxyguanosine

[0326] A reaction was conducted as described in Example 43 except that8-azaguanine (Tokyo Kasei) was used in place of 4-azabenzimidazole. HPLCanalysis for the post-reaction solution as described in Example 44showed a peak of 8-aza-2′-deoxyguanosine. A reaction inversion rate wascalculated to be 36.1% after determining the concentration of8-aza-2′-deoxyguanosine in the post-reaction solution.

Example 46

[0327] Preparation of 2-chloro-2 -deoxyadenosine (Cladribine)

[0328] A reaction was conducted as described in Example 43 except that2-chloro-4-aminopurine was used in place of 4-azabenzimidazole. HPLCanalysis for the post-reaction solution under the conditions describedbelow showed a peak of the title compound. A reaction inversion rate wascalculated to be 96% after determining the concentration of2-chloro-2′-deoxyadenosine in the post-reaction solution.

[0329] HPLC Analysis Conditions

[0330] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0331] Column temperature: 40° C.

[0332] Pump flow rate: 1.0 mL/min

[0333] Detection: UV 254 nm

[0334] Eluent: 25 mM potassium dihydrogen phosphate methanol=875:125(V/V)

Example 47

[0335] Preparation of1-(β-D-ribofuranos-1-yl)-1,3,4-triazole-3-carboxamide (Ribavirine)

[0336] A reaction was conducted as described in Example 43 except that1,2,4-tosyazole-3-carboxamide was used in place of 4-azabenzimidazole.HPLC analysis for the post-reaction solution under the conditionsdescribed below showed a peak of the title compound. A reactioninversion rate was calculated to be 69% after determining theconcentration of 1-(β-D-ribofuranos-1-yl)-1,3,4-triazole-3-carboxamidein the post-reaction solution.

[0337] HPLC Analysis Conditions

[0338] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0339] Column temperature: 40° C.

[0340] Pump flow rate: 1.0 mL/min

[0341] Detection: UV 210 nm

[0342] Eluent: 25 mM potassium dihydrogen phosphate

Example 48

[0343] Preparation of1-(β-D-ribofuranos-1-yl)-5-aminoimidazole-4-carboxamide (Acadesine)

[0344] A reaction was conducted as described in Example 43 except that5-aminoimidazole-4-carboxamide was used in place of 4-azabenzimidazole.HPLC analysis for the post-reaction solution under the conditionsdescribed below showed a peak of the title compound. A reactioninversion rate was calculated to be 46% after determining theconcentration of 1-(β-D-ribofuranos-1-yl)-5-aminoimidazole-4-carboxamidein the post-reaction solution.

[0345] HPLC Analysis Conditions

[0346] Column: Develosil ODS-MG-5, 250×4.6 mm I.D.

[0347] Column temperature: 40° C.

[0348] Pump flow rate: 1.0 mL/min

[0349] Detection: UV 254 nm

[0350] Eluent: 25 mM potassium dihydrogen phosphate methanol=93:7 (V/V)

Example 49

[0351] Preparation of 2′-deoxyguanosine

[0352] To 20 g of purified water were added 3.22 g of2-deoxyribose-1-phosphate di(monocyclohexylammonium) salt prepared inExample 24 (7.72 mmol), 1.11 g of guanine (7.34 mmol) and 0.67 g ofmagnesium hydroxide (11.48 mmol). The reaction mixture was adjusted topH 9 with a 20% aqueous solution of sodium hydroxide. To the mixture wasadded 0.1 mL of the above enzyme solution (0.1 mL), and the mixture wasreacted with stirring at 50° C. for 8 hours. HPLC analysis for thereaction mixture after 8 hours indicated that the desired2′-deoxyguanosine was provided with a reaction yield of 99%.

Example 50

[0353] Preparation of 2′-deoxyadenosine

[0354] To 20 g of purified water were added 3.22 g of2-deoxyribose-1-phosphate di(monocyclohexylammonium) salt prepared inExample 24 (7.72 mmol), 1.01 g of adenine (7.47 mmol) and 0.67 g ofmagnesium hydroxide (11.48 mmol). The reaction mixture was adjusted topH 8.6 with a 20% aqueous solution of sodium hydroxide. To the mixturewas added 0.1 mL of the above enzyme solution (0.1 mL), and the mixturewas reacted with stirring at 50° C. for 3 hours. HPLC analysis for thereaction mixture after 8 hours indicated that the desired2′-deoxyadenosine was provided with a reaction yield of 99%.

INDUSTRIAL APPLICABILITY

[0355] As described above, this invention is quite useful as an anomerselective process for producing a 1-phosphorylated saccharide derivativeor a nucleoside and may be expected to used in a variety ofapplications.

1 2 1 30 DNA Artificial Sequence primer 1 gtgaattcac aaaaaggataaaacaatggc 30 2 29 DNA Artificial Sequence primer 2 tcgaagcttgcgaaacacaa ttactcttt 29

What is claimed is:
 1. A process for selectively preparing either α or βisomer of a 1-phosphorylated saccharide derivative monomer comprisingthe steps of phosphorolyzing and isomerizing an anomer mixture of a1-phosphorylated saccharide derivative to give α and β isomers of the1-phosphorylated saccharide derivative monomer and selectivelycrystallizing one of these isomers to displace the equilibrium betweenthese anomers.
 2. A process for selectively preparing either a or βisomer of a 1-phosphorylated saccharide derivative monomer comprisingthe steps of phosphorolyzing and isomerizing an anomer mixture of a1-phosphorylated saccharide derivative represented by formula (1):

where R¹ and R² independently represents hydrogen, methyl, protectedhydroxymethyl or protected carboxyl; R³ represents acyl; R⁴ represents aprotective group for hydroxy; x represents halogen, alkoxy or alkylthio;W represents oxygen or sulfur; Z represents oxygen, sulfur or optionallysubstituted carbon; m represents an integer of 1 to 3; n represents 0 or1; p and q represents an integer of 0 to 4; and r represents 0 or 1;provided that p, q, r and n meet the conditions of p+r≦n+1 andq≦2×(n+1)−2×(p+r) when Z is oxygen or sulfur and of p+r≦n+2 andq≦2×(n+2)−2×(p+r) when Z is carbon, to give α and β isomers of the1-phosphorylated saccharide derivative monomer and selectivelycrystallizing one of these isomers to displace the equilibrium betweenthese anomers:
 3. A process for preparing a 1-phosphorylated saccharidederivative monomer represented by formula (3):

wherein R¹ and R² independently represents hydrogen, methyl,hydroxymethyl or carboxyl; R³ represents hydrogen or acyl; and X, W, Z,n, p, q and r are as defined above, comprising the steps ofphosphorolyzing and isomerizing an anomer mixture of a 1-phosphorylatedsaccharide derivative represented by formula (1):

wherein R¹, R², R³, R⁴, X, W, Z, m, n, p, q and r are as defined inclaim 2, to give α and β isomers of the 1-phosphorylated saccharidederivative monomer; selectively crystallizing one of these isomers todisplace the equilibrium between these anomers; and then removing theprotective group represented by R⁴.
 4. A trimer, dimer or monomer of a1-phosphorylated saccharide derivative represented by formula (4):

wherein R¹ and R² independently represents hydrogen, methyl,hydroxymethyl protected with substituted benzoyl or protected carboxyl;R⁴ represents hydrogen or a protective group for hydroxy; and R³, X, W,Z, m, n, p, q and r are as defined in claim 2, or salts thereof.
 5. A1-phosphorylated saccharide derivative monomer represented by formula(5):

wherein p and q represents an integer of 0 to 3; r represents 0 or 1;and R¹, R², R³, R⁴, X, W and Z are as defined in claim 2; provided thatp, q and r meet the conditions of p+q+r≦3 when Z is oxygen or sulfur andof p+q+r≦5 when Z is carbon, or salts thereof.
 6. A 1-phosphorylatedsaccharide derivative monomer represented by formula (6):

wherein R¹ and R² independently represents hydrogen, methyl,hydroxymethyl or carboxy; and R³, X, W, Z, n, p, q and r are as definedin claim 2 other than natural products, or salts thereof.
 7. The1-phosphorylated saccharide derivative monomer as claimed in claim 6wherein n=1 in formula (6), or its salt.
 8. The 1-phosphorylatedsaccharide derivative monomer as claimed in claim 7 wherein R¹ ishydroxymethyl; R² is hydrogen; p and r are 0; and X is fluorine, or itssalt.
 9. A process for preparing a 1-phosphorylated sacchariderepresented by formula (20):

wherein R¹¹ represents protected hydroxymethyl and R¹⁴ represents aprotective group for hydroxy, comprising the steps of treating acompound represented by formula (18):

wherein R¹¹ and R¹⁴ are as defined above, with phosphoric acid in thepresence of a base to give an anomer mixture of a 1-phosphorylatedsaccharide derivative represented by formula (19):

wherein R¹¹ and R¹⁴ are as defined above and m is as defined in claim 2;phosphorolyzing and isomerizing the mixture; and displacing theequilibrium between the anomer isomers by selectively crystallizing anα-isomer formed.
 10. A process for preparing2-deoxy-α-D-ribose-1-phosphate comprising the steps of treating acompound represented by formula (18):

wherein R¹¹ represents protected hydroxymethyl and R¹⁴ represents aprotective group for hydroxy, with phosphoric acid in the presence of abase to give an anomer mixture of a 1-phosphorylated saccharidederivative represented by formula (19):

wherein R¹¹ and R¹⁴ are as defined above and m is as defined in claim 2;phosphorolyzing and isomerizing the mixture; displacing the equilibriumbetween the anomer isomers by selectively crystallizing an α-isomerformed to give the α-isomer; and then removing the protective group. 11.A process for preparing a nucleoside represented by formula (8):

wherein B is a base independently selected from the group consisting ofpyrimidine, purine, azapurine and deazapurine optionally substituted byhalogen, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino,alkylamino, hydroxy, hydroxyamino, aminoxy, alkoxy, mercapto,alkylmercapto, aryl, aryloxy or cyano; and R¹, R², R³, X, W, Z, n, p, qand r are as defined for formula (3) in claim 3, comprising the firstprocedure for preparing the 1-phosphorylated saccharide derivativemonomer as claimed in claim 3; and the second procedure of conducting anexchange reaction of the phosphate group in the 1-phosphorylatedsaccharide derivative obtained in the first procedure with a base by theaction of a nucleoside phosphorylase.
 12. A process for preparing anucleoside represented by formula (8):

wherein B is as defined for formula (8) in claim 11; and R¹, R², R³, R⁴,X, W, Z, n, p, q and r are as defined for formula (6) in claim 6,comprising an exchange reaction of the phosphate group in the1-phosphorylated saccharide derivative monomer in as claimed in claim 6with a base by the action of a nucleoside phosphorylase.
 13. A processfor preparing a nucleoside represented by formula (10):

wherein B is as defined for formula (8) in claim 11; and R¹, R², R³, R⁴,X, W, Z, n, p, q and r are as defined for formula (7) in claim 7,comprising an exchange reaction of the phosphate group in the1-phosphorylated saccharide derivative monomer as claimed in claim 7with a base by the action of a nucleoside phosphorylase.
 14. The processfor preparing a nucleoside as claimed in claim 13 wherein R¹ ishydroxymethyl, R² is hydrogen, p and r are 0, and X is fluorine.
 15. Aprocess for preparing a nucleoside represented by formula (21):

wherein B is as defined for formula (8) in claim 11, comprising thefirst procedure of preparing 2-deoxy-α-D-ribose-1-phosphate as claimedin claim 14; and the second procedure of conducting an exchange reactionof the phosphate group in the 1-phosphorylated saccharide derivativeobtained in the first procedure with a base by the action of anucleoside phosphorylase.
 16. The process for preparing a nucleoside asclaimed in any of claims 11 to 15 wherein the nucleoside phosphorylaseis at least one enzyme selected from the group consisting of purinenucleoside phosphorylase (EC2.4.2.1), guanosine nucleoside phosphorylase(EC2.4.2.15), pyrimidine nucleoside phosphorylase (EC2.4.2.2), uridinenucleoside phosphorylase (EC2.4.2.3), thymidine nucleoside phosphorylase(EC2.4.2.4) and deoxyuridine nucleoside phosphorylase (EC2.4.2.23). 17.The process for preparing a nucleoside as claimed in any of claims 11 to15 wherein the nucleoside phosphorylase activity is replaced with amicroorganism expressing at least one nucleoside phosphorylase selectedfrom the group consisting of purine nucleoside phosphorylase(EC2.4.2.1), guanosine nucleoside phosphorylase (EC2.4.2.15), pyrimidinenucleoside phosphorylase (EC2.4.2.2), uridine nucleoside phosphorylase(EC2.4.2.3), thymidine nucleoside phosphorylase (EC2.4.2.4) anddeoxyuridine nucleoside phosphorylase (EC2.4.2.23).
 18. The process forpreparing a nucleoside as claimed in any of claims 11 to 15 wherein ametal cation capable of forming a water-insoluble salt with a phosphateion is present in the reaction solution during the exchange reaction ofa phosphate group in the 1-phosphorylated saccharide derivative monomerwith a base by the action of a nucleoside phosphorylase.
 19. The processfor preparing a nucleoside as claimed in claim 18 wherein the metalcation capable of forming a water-insoluble salt with the phosphate ionis at least one metal cation selected from the group consisting ofcalcium, barium, aluminum and magnesium ions.
 20. The process forpreparing a nucleoside as claimed in claim 8 wherein the nucleoside is anatural nucleoside.
 21. A synthetic nucleoside represented by formula(11):

wherein B, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are as defined forformulas (8) in claim 11 or its salt, excluding trifluorothymidine,ribavirin, orotidine, uracil arabinoside, adenine arabinoside,2-methyl-adenine arabinoside, 2-chloro-hypoxanthine arabinoside,thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosinearabinoside, guanine arabinoside, thymine arabinoside, enocitabine,gemcitabine, azidothymidine, idoxuridine, dideoxyadenosine,dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine,thiadideoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine,tegafur, doxifluridine, bredinin, nebularine, allopurinol uracil,5-fluorouracil, 2′-aminouridine, 2′-aminoadenosine, 2′-aminoguanidine,2-chloro-2′-aminoinosine, DMDC and FMDC.
 22. A synthetic nucleosiderepresented by formula (12):

wherein B, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are as defined forformulas (8) in claim 11 or its salt, excluding trifluorothymidine,ribavirin, orotidine, uracil arabinoside, adenine arabinoside,2-methyl-adenine arabinoside, 2-chloro-hypoxanthine arabinoside,thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosinearabinoside, guanine arabinoside, thymine arabinoside, enocitabine,gemcitabine, azidothymidine, idoxuridine, dideoxyadenosine,dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine,thiadideoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine,tegafur, doxifluridine, bredinin, nebularine, allopurinol uracil,5-fluorouracil, 2′-aminouridine, 2′-aminoadenosine, 2′-aminoguanidine,2-chloro-2′-aminoinosine, DMDC and FMDC.
 23. A nucleoside represented byformula (13):

wherein B, R¹, R², R³, R⁴, X, W, Z, n, p, q and r are as defined forformulas (8) in claim 11 or its salt, excluding trifluorothymidine,ribavirin, orotidine, uracil arabinoside, adenine arabinoside,2-methyl-adenine arabinoside, 2-chloro-hypoxanthine arabinoside,thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosinearabinoside, guanine arabinoside, thymine arabinoside, enocitabine,gemcitabine, azidothymidine, idoxuridine, dideoxyadenosine,dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine,thiadideoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine,tegafur, doxifluridine, bredinin, nebularine, allopurinol uracil,5-fluorouracil, 2′-aminouridine, 2′-aminoadenosine, 2′-aminoguanidine,2-chloro-2′-aminoinosine, DMDC and FMDC.
 24. A 1-phosphorylatedsaccharide represented by formula (20):

wherein R¹¹ represents protected hydroxymethyl; and R¹⁴ represents aprotective group for hydroxy, or its salt.